Polypeptides

ABSTRACT

The present disclosure relates to a class of engineered polypeptides having a binding affinity for the neonatal Fc receptor (in the following referred to as FcRn), and provides an FcRn binding polypeptide comprising the sequence EX 2  X 3  X 4  AX 6  X 7  EIRWLPNL X 16 X 17  X 18  QRX 21  AFIX 25  X 26 LX 28  X 29  (SEQ ID NO:1075). The present disclosure also relates to the use of such an FcRn binding polypeptide as an agent for modifying pharmacokinetic and pharmacodynamic properties and as a therapeutic agent.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is Continuation of U.S. application Ser. No. 15/842,178filed Dec. 14, 2017 which is a Continuation of U.S. application Ser. No.14/776,319 filed Sep. 14, 2015, now U.S. Pat. No. 9,975,943, which is aU.S. National Stage Application of PCT/EP2014/055299 filed Mar. 17,2014, which claims priority from European Patent Application No.:13159500.1, filed Mar. 15, 2013, and U.S. Provisional Application Ser.No. 61/787,305 filed Mar. 15, 2013, all of which are incorporated byreference in their entirety.

TECHNICAL FIELD OF THE INVENTION

The present disclosure relates to a class of engineered polypeptideshaving a binding affinity for the neonatal Fc receptor (in the followingreferred to as FcRn). The present disclosure also relates to the use ofsuch an FcRn binding polypeptide as an agent for modifyingpharmacokinetic and pharmacodynamic properties of a biomolecule, e.g. apharmaceutical, and as a therapeutic agent.

BACKGROUND

The neonatal Fc receptor (FcRn) is a heterodimeric protein consisting ofa transmembrane MHC class I-like heavy chain (FcRn α-chain) and theβ2-microglobulin light chain, the latter also forming a part of MHCclass I molecules (Simister and Mostov (1989) Nature 337:184-7;Burmeister et al. (1994) Nature 372:379-83).

FcRn is predominantly located in endosomes and is able to bind to serumalbumin and immunoglobulin G (IgG) at pH≥6.5 and release them at pH≥7.0(reviewed in Roopenian (2007) Nat Rev Immunol 7:715-25).

FcRn carries out several distinct tasks in mammals (reviewed inRoopenian, supra). FcRn is involved in recycling of endocytosed IgG andserum albumin, thus avoiding their degradation in the lysosome, givingthem longer half-life and higher availability in the blood than otherserum proteins. When IgG, serum albumin and other serum proteins arepassively pinocytosed by cells in contact with blood, the pH becomesgradually lower in the formed endosomes, which permits the binding ofIgG and serum albumin to FcRn. The receptor is then, together with itsbound ligand, transported via recycling endosomes back to the plasmamembrane. After returning to the plasma membrane, the pH increases toabove 7, at which point the bound ligand is released.

FcRn is also recognized for its ability to transport IgG over barrierssuch as the placenta, the upper airway epithelium, the blood-brainbarrier and the proximal small intestine.

In mammals, the properties of FcRn are used to transcytose IgG from amother to a fetus via the placenta, and to transcytose IgG from amother's milk to the blood stream of an infant in the proximal smallintestine.

The expression pattern of FcRn differs between species. However, FcRn iswidely expressed by cells in the blood brain barrier, upper airwayepithelium, kidneys and vascular endothelia, and by antigen presentingcells as well as by other cells of hematopoietic origin in most species(reviewed in Roopenian (2007), supra).

Antibodies and peptides with affinity towards FcRn (Liu et al. (2007) JImmunol 179:2999-3011, Mezo et al. (2008) Proc Natl Acad Sci USA105:2337-42) and β2-microglobulin (Getman and Balthasar (2005) J PharmSci 94:718-29) have been developed with a view to inhibit the bindingbetween endogenous IgG and FcRn. Another approach has been to mutate theFc region of the IgG to get a higher affinity for FcRn (Petkova et al.(2006) Int Immunol 18:1759-69, Vaccaro et al. (2005) Nat Biotechnol23:1283-8).

Fusion to the Fc domain or to albumin is a widely used strategy toincrease the in vivo half-life of proteins. However, the large size ofsuch fusion proteins adversly affects tissue penetration and reduces thespecificity to the fusion partner (Valles et al. (2011) J InterferonCytokine Res 32:178-184). On the other hand, mutations have been made inthe Fc fragment of antibodies administered to non human primates toprolong half-life (Hinton et al. (2004) J Biol Chem 279:6213-6).However, this approach is only limited in use to therapeutic antibodies,and cannot be extrapolated to other therapeutic proteins unless theproteins in question are fused to Fc fragments, which also results inlarge size molecules. A number of chemical and recombinant methods havebeen devised to improve protein half-life, such as PEGylation andgenetic fusions of the protein to the Fc domain of IgG or albumin(reviewed in Schellenberger et al. (2009) Nat Biotechnol 21:1186-1190).PEGylation of proteins has been reported to decrease their potency andcontribute to their immunoreactivity.

Fc-fusion proteins have also been used for oral and pulmonary deliverymediated by the FcRn (Low et al., (2005) Human reproduction July;20(7):1805-13), however similar problems relating to tissue penetrationand reduced specificity remain, due to the size of the fusion molecules.

Hence, there is large need in the field for the continued provision ofmolecules with high affinity for FcRn. In particular, small bindingmolecules are needed that, when present as a fusion partner, do notadversely affect the properties of the molecules they are fused to anddo not contribute to immunoreactivity.

SUMMARY OF THE INVENTION

It is an object of the present disclosure to provide new FcRn bindingagents for use in modifying pharmacokinetic and/or pharmacodynamicproperties of a biomolecule, e.g. a pharmaceutical.

It is also an object of the present disclosure to provide new FcRnbinding agents for use as therapeutic agents in their own right, aloneor as combination treatment.

It is an object of the present disclosure to provide a molecule allowingfor efficient targeting of FcRn, while alleviating the above-mentionedand other drawbacks of current therapies.

These and other objects which are evident to the skilled person from thepresent disclosure are met by different aspects of the invention asclaimed in the appended claims and as generally disclosed herein.

Thus, in the first aspect of the disclosure, there is provided aneonatal Fc receptor (FcRn) binding polypeptide, comprising an FcRnbinding motif, BM, which motif consists of the amino acid sequence

(SEQ ID NO: 1075) EX₂ X₃ X₄ AX₆ X₇ EIR WLPNLX₁₆ X₁₇ X₁₈ QR X₂₁AFIX₂₅ X₂₆LX₂₈ X₂₉wherein, independently from each other,

X₂ is selected from A, D, E, F, H, I, K, L, N, Q, R, S, T, V, W and Y;

X₃ is selected from A, D, E, F, G, H, I, K, L, M, N, Q, R, S, T, V, Wand Y;

X₄ is selected from A, D, E, F, G, H, I, K, L, N, Q, R, S, T, V, W andY;

X₆ is selected from A, D, E, F, G, H, I, K, L, N, Q, R, S, T, V, W andY;

X₇ is selected from A, F, H, I, K, L, N, Q, R, S, T, V, W and Y;

X₁₆ is selected from N and T;

X₁₇ is selected from F, W and Y;

X₁₈ is selected from A, D, E and N;

X₂₁ is selected from A, S, V and W;

X₂₅ is selected from A, D, E, F, G, H, I, K, L, N, Q, R, S, T, V, W andY;

X₂₆ is selected from K and S,

X₂₈ is selected from A, D, E, F, H, I, K, L, N, Q, R, S, T, V, W and Y;and

X₂₉ is selected from D and R.

The above definition of a class of sequence related, FcRn bindingpolypeptides is based on a statistical analysis of a number of randompolypeptide variants of a parent scaffold, that were selected for theirinteraction with FcRn in several different selection experiments. Theidentified FcRn binding motif, or “BM”, corresponds to the targetbinding region of the parent scaffold, which region constitutes twoalpha helices within a three-helical bundle protein domain. In theparent scaffold, the varied amino acid residues of the two BM helicesconstitute a binding surface for interaction with the constant Fc partof antibodies. In the present disclosure, the random variation ofbinding surface residues and subsequent selection of variants havereplaced the Fc interaction capacity with a capacity for interactionwith FcRn.

In one embodiment of said FcRn binding polypeptide, the BM consists ofthe amino acid sequence

(SEQ ID NO: 1076) EX₂ X₃ X₄ AX₆ X₇ EIR WLPNLTX₁₇ X₁₈ QR X₂₁ AFIX₂₅KLX₂₈ Dwherein, independently from each other,

X₂ is selected from A, D, E, F, H, I, K, L, N, Q, R, S, T, V, W and Y;

X₃ is selected from A, D, E, F, G, H, I, K, L, M, N, Q, R, S, T, V, Wand Y;

X₄ is selected from A, D, E, F, G, H, I, K, L, N, Q, R, S, T, V, W andY;

X₆ is selected from A, D, E, F, G, H, I, K, L, N, Q, R, S, T, V, W andY;

X₇ is selected from A, F, H, I, K, L, N, Q, R, S, T, V, W and Y;

X₁₇ is selected from F, W and Y;

X₁₈ is selected from A, D, E and N;

X₂₁ is selected from A, S, V and W;

X₂₅ is selected from A, D, E, F, G, H, I, K, L, N, Q, R, S, T, V, W andY; and

X₂₈ is selected from A, D, E, F, H, I, K, L, N, Q, R, S, T, V, W and Y.

In another embodiment of the first aspect of the disclosure, saidneonatal Fc receptor (FcRn) binding polypeptide comprises an FcRnbinding motif, BM, which motif consists of the amino acid sequence

(SEQ ID NO: 1075) EX₂ X₃ X₄ AX₆ X₇ EIR WLPNLX₁₆X₁₇ X₁₈ QR X₂₁AFIX₂₅ X₂₆LX₂₈ X₂₉wherein, independently from each other,

X₂ is selected from A, D, E, F, H, I, K, L, N, Q, R, S, T, V, W and Y;

X₃ is selected from A, D, E, F, G, H, I, K, L, M, N, Q, R, S, T, V, Wand Y;

X₄ is selected from A, D, E, F, G, H, I, K, L, N, Q, R, S, T, V, W andY;

X₆ is selected from A, E, F, G, H, I, K, Q, R, S and V;

X₇ is selected from A, F, H, K, N, Q, R, S and V;

X₁₆ is selected from N and T;

X₁₇ is selected from F, W and Y;

X₁₈ is selected from A, D, E and N;

X₂₁ is selected from A, S, V and W;

X₂₅ is selected from D, E, G, H, I, K, L, N, Q, R, S, T, V, W and Y;

X₂₆ is selected from K and S,

X₂₈ is selected from A, D, E, F, H, I, K, L, N, Q, R, S, T, V, W and Y;and

X₂₉ is selected from D and R.

In another embodiment of the first aspect, there is provided an FcRnbinding polypeptide, wherein, independently from each other,

X₂ is selected from A, D, E, F, H, I, K, L, N, Q, R, S, T, V, W and Y;

X₃ is selected from A, D, E, F, H, I, K, L, M, N, Q, R, S, T, V, W andY;

X₄ is selected from A, D, E, F, H, I, K, L, N, Q, R, S, T, V, W and Y;

X₆ is selected from A, E, F, G, H, I, K, Q, R and S,

X₇ is selected from A, F, H, K, N, Q, R, S and V;

X₁₆ is selected from N and T;

X₁₇ is selected from F and Y;

X₁₈ is D;

X₂₁ is V;

X₂₅ is selected from D, E, H, I, K, L, N, Q, R, S, T, V, W and Y;

X₂₆ is selected from K and S,

X₂₈ is selected from A, D, E, F, H, I, K, L, N, Q, R, S, T, V and W and.

X₂₉ is selected from D and R.

In another embodiment of the first aspect, the BM consists of an aminoacid sequence selected from

i) (SEQ ID NO: 1076) EX₂ X₃ X₄ AX₆ HEIR WLPNLTX₁₇ X₁₈ QR X₂₁ AFIX₂₅KLX₂₈ Dwherein, independently from each other,

X₂ is selected from A, D, E, F, H, I, K, L, N, Q, R, S, T, V, W and Y;

X₃ is selected from A, D, E, G, H, K, L, M, N, Q, R, S, T, V and Y;

X₄ is selected from A, D, E, F, G, I, K, L, N, Q, R, S, T, V and Y;

X₆ is selected from A, G, K, R, S and V;

X₁₇ is selected from F, W and Y;

X₁₈ is selected from A, D, E and N;

X₂₁ is selected from A, S, V and W;

X₂₅ is selected from D, G, H, K, L, N, R, V and W;

X₂₈ is selected from A, D, E, H, K, L, N, Q, R, S, T, W and Y; and

-   -   ii) an amino acid sequence which has at least 96% identity to        said sequence.

In yet another embodiment of said aspect, the BM in sequence i) consistsof an amino acid sequence selected from

(SEQ ID NO: 1076) EX₂ X₃ X₄ AX₆ HEIR WLPNLTX₁₇ X₁₈ QR X₂₁ AFIX₂₅ KLX₂₈ Dwherein, independently from each other,

X₂ is selected from A, D, E, F, N, Q, R, S and W;

X₃ is selected from D, E, G, H, K, M, N, Q, S and T;

X₄ is selected from A, D, E, G, N, Q, R, S, T, V and Y;

X₆ is selected from A, G, S and V;

X₁₇ is selected from F, W and Y;

X₁₈ is selected from A, D, E and N;

X₂₁ is selected from A, S, V and W;

X₂₅ is selected from D, G, H, K, L, N, R and V; and

X₂₈ is selected from A, E, H, L, N, Q, R, S, T, W and Y.

As the skilled person will realize, the function of any polypeptide,including the FcRn binding capacity of the polypeptide of the presentdisclosure, is dependent on the tertiary structure of the polypeptide.It is therefore possible to make minor changes to the sequence of aminoacids in a polypeptide without affecting the function thereof. Thus, thedisclosure encompasses modified variants of the FcRn bindingpolypeptide, which are such that the FcRn binding characteristics areretained.

Therefore, as described above, also encompassed by the presentdisclosure is a FcRn binding polypeptide comprising an amino acidsequence with 96% or greater identity to a polypeptide as defined in i).

In some embodiments, such changes may be made in all positions of thesequences of the FcRn binding polypeptide as disclosed herein. In otherembodiments, such changes may be made only in the non-variablepositions, also denoted as scaffold amino acid residues. In such cases,changes are not allowed in the variable positions, i.e. positionsdenoted with an “X” in sequence i). For example, it is possible that anamino acid residue belonging to a certain functional grouping of aminoacid residues (e.g. hydrophobic, hydrophilic, polar etc) could beexchanged for another amino acid residue from the same functional group.

The term “% identity”, as used throughout the specification, may forexample be calculated as follows. The query sequence is aligned to thetarget sequence using the CLUSTAL W algorithm (Thompson et al. (1994)Nucleic Acids Research 22:4673-4680). A comparison is made over thewindow corresponding to the shortest of the aligned sequences. Theshortest of the aligned sequences may in some instances be the targetsequence. In other instances, the query sequence may constitute theshortest of the aligned sequences. The amino acid residues at eachposition are compared, and the percentage of positions in the querysequence that have identical correspondences in the target sequence isreported as % identity.

Below follows a list of embodiments which further specify amino acidresidue X_(n), wherein n is an integer which denotes the position ofsaid residue within the polypeptide described herein. To clarify, incases where the BM comprised in the polypeptide may consist of either agiven amino acid sequence or an amino acid sequence with at least agiven % identity to said given amino acid sequence, the X_(n) as usedherein refers to an amino acid residue in said given amino acidsequence. For example, when applicable, X_(n) refers to an amino acidresidue in sequence i) above.

In one embodiment, X₂ is selected from A, D, E, F, I, L, N, Q, R, S, T,V, W and Y.

In one embodiment, X₂ is selected from A, D, F, I, L, N, Q, R, S, T, V,W and Y.

In one embodiment, X₂ is selected from A, D, F, I, L, N, Q, R, S, V and

W.

In one embodiment, X₂ is selected from A, I, L, N, Q, R, S, T, V, W andY.

In one embodiment, X₂ is selected from A, I, L, N, Q, S, T, V and W.

In one embodiment, X₂ is selected from A, I, L, N, Q, V and W.

In one embodiment, X₂ is selected from A, I, L, Q, V and W.

In one embodiment, X₂ is selected from A, I, L and Q.

In one embodiment, X₂ is selected from I, L and Q.

In one embodiment, X₂ is selected from I and Q.

In one embodiment, X₂ is I.

In one embodiment, X₂ is Q.

In one embodiment, X₃ is selected from A, D, E, G, H, K, L, M, N, Q, R,S, T, V and Y.

In one embodiment, X₃ is selected from A, D, E, H, K, L, M, N, Q, R, S,T, V and Y.

In one embodiment, X₃ is selected from A, D, E, G, H, K, L, M, N, Q, R,S and T.

In one embodiment, X₃ is selected from A, D, E, G, H, K, M, N, Q, S andT.

In one embodiment, X₃ is selected from A, D, E, G, H, M, N, Q, S and T.

In one embodiment, X₃ is selected from A, D, E, K, N, Q, S and T.

In one embodiment, X₃ is selected from A, D, E, K, Q, and T.

In one embodiment, X₃ is selected from A, D, E, Q and T.

In one embodiment, X₃ is selected from D, E and T.

In one embodiment, X₃ is selected from D and E.

In one embodiment, X₃ is D.

In one embodiment, X₃ is E.

In one embodiment, X₄ is selected from A, D, E, F, G, I, K, L, N, Q, R,S, T, V and Y.

In one embodiment, X₄ is selected from A, D, E, G, N, Q, R, S, T and V.

In one embodiment, X₄ is selected from A, D, E, F, I, K, L, N, Q, R, S,T and V.

In one embodiment, X₄ is selected from A, D, E, I, K, N, Q, R, S and T.

In one embodiment, X₄ is selected from A, D, E, I, K, Q, S and T.

In one embodiment, X₄ is selected from A, D, I, K, Q and S.

In one embodiment, X₄ is selected from A, D, E, K and S.

In one embodiment, X₄ is selected from A, D, K and S.

In one embodiment, X₄ is selected from A, D, E and K.

In one embodiment, X₄ is selected from A, D and K.

In one embodiment, X₄ is selected from A and D.

In one embodiment, X₄ is selected from A and E.

In one embodiment, X₄ is A.

In one embodiment, X₄ is D.

In one embodiment, X₄ is E.

In one embodiment, X₆ is selected from A, G, K, Q, R, S and V.

In one embodiment, X₆ is selected from A, G, K, R, S and V.

In one embodiment, X₆ is selected from A, G, K, R and S.

In one embodiment, X₆ is selected from A, G, K, S and V.

In one embodiment, X₆ is selected from A, G, K and V.

In one embodiment, X₆ is selected from A, G, K and S.

In one embodiment, X₆ is selected from A, G and K.

In one embodiment, X₆ is selected from A, G and V.

In one embodiment, X₆ is selected from A and G.

In one embodiment, X₆ is A.

In one embodiment, X₆ is G.

In one embodiment, X₇ is selected from A and H.

In one embodiment, X₇ is H.

In one embodiment, X₁₆ is T.

In one embodiment, X₁₆ is N.

In one embodiment, X₁₇ is selected from F and Y.

In one embodiment, X₁₇ is F.

In one embodiment, X₁₈ is selected from A, D and E.

In one embodiment, X₁₈ is selected from A and D.

In one embodiment, X₁₈ is D.

In one embodiment, X₂₁ is selected from V and W.

In one embodiment, X₂₁ is V.

In one embodiment, X₂₅ is selected from D, E, G, H, K, L, N, Q, R, V andW.

In one embodiment, X₂₅ is selected from D, G, H, K, L, N, R, V and W.

In one embodiment, X₂₅ is selected from D, G, H, K, L, N, R and V.

In one embodiment, X₂₅ is selected from H, L, R, V and W.

In one embodiment, X₂₅ is selected from H, R, V and W.

In one embodiment, X₂₅ is selected from H, R and V.

In one embodiment, X₂₅ is selected from H, L and R.

In one embodiment, X₂₅ is selected from H and R.

In one embodiment, X₂₅ is selected from H and V.

In one embodiment, X₂₅ is H.

In one embodiment, X₂₆ is K.

In one embodiment, X₂₆ is S.

In one embodiment, X₂₈ is selected from A, D, E, H, K, L, N, Q, R, S, T,W and Y.

In one embodiment, X₂₈ is selected from A, D, E, K, L, N, Q, R, S, T, Wand Y.

In one embodiment, X₂₈ is selected from A, D, E, L, R, S, T, W and Y.

In one embodiment, X₂₈ is selected from A, D, K, L, N, Q, R, S, T and W.

In one embodiment, X₂₈ is selected from A, D and R.

In one embodiment, X₂₈ is selected from A and R.

In one embodiment, X₂₈ is selected from D and R.

In one embodiment, X₂₈ is A.

In one embodiment, X₂₈ is R.

In one embodiment, X₂₉ is D.

In one embodiment, X₂₉ is R.

In one embodiment, X₆X₇ is selected from AH and GH.

In one embodiment, X₆X₇ is AH.

In one embodiment, X₆X₇ is GH.

In one embodiment, X₁₇X₁₈ is selected from FD and YD.

In one embodiment, X₁₇X₁₈ is FD.

In a more specific embodiment defining a sub-class of the FcRn bindingpolypeptide, the sequence fulfills at least three of the six conditionsI-VI:

-   -   I. X₆ is selected from A, G, K and S, such as in particular A;    -   II. X₇ is H,    -   III. X₁₇ is selected from F and Y, such as in particular F;    -   IV. X₁₈ is D;    -   V. X₂₁ is selected from V and W, such as in particular V;    -   VI. X₂₅ is selected from H and R, such as in particular H.

In some examples of an FcRn binding polypeptide according to the firstaspect, said sequence fulfills at least four of the six conditions I-VI.More specifically, the sequence may fulfill at least five of the sixconditions I-VI, such as all of the six conditions I-VI.

As described in detail in the experimental section to follow, theselection of FcRn binding polypeptide variants has led to theidentification of a number of individual FcRn binding motif (BM)sequences. These sequences constitute individual embodiments accordingto this aspect. The sequences of individual FcRn binding motifs arepresented in FIGS. 1A-1MM and as SEQ ID NO:1-353. Hence, in oneembodiment of the FcRn binding polypeptide according to this aspect, thesequence is selected from the group consisting of SEQ ID NO:1-353. Inone embodiment, the sequence is selected from the group consisting ofSEQ ID NO:1-15, SEQ ID NO:17-140 and SEQ ID NO:353. In one embodiment,the sequence is selected from the group consisting of SEQ ID NO:1-2 andSEQ ID NO:17-140. In one embodiment, the sequence is selected from thegroup consisting of SEQ ID NO:1-2, SEQ ID NO:17-92, SEQ ID NO:94-103,SEQ ID NO:105-125 and SEQ ID NO:127-140. In one embodiment, the sequenceis selected from the group consisting of SEQ ID NO:1-8, SEQ ID NO:13 SEQID NO:19-20, SEQ ID NO:23, SEQ ID NO:28, SEQ ID NO:41, SEQ ID NO:44, SEQID NO:65, SEQ ID NO:70, SEQ ID NO:73, SEQ ID NO:75-77 and SEQ ID NO:353.In another embodiment, the sequence is selected from the groupconsisting of SEQ ID NO:1, SEQ ID NO:23, SEQ ID NO:28, SEQ ID NO:41, SEQID NO:44, SEQ ID NO:65, SEQ ID NO:73 and SEQ ID NO:75-77. In yet anotherembodiment, the sequence is selected from SEQ ID NO:1, SEQ ID NO:23, SEQID NO:44, SEQ ID NO:65, SEQ ID NO:75 and SEQ ID NO:77. In oneembodiment, the sequence is selected from SEQ ID NO:1, SEQ ID NO:23 andSEQ ID NO:75. In one embodiment, the sequence is SEQ ID NO:1.

In some embodiments of the present disclosure, the BM as defined above“forms part of” a three-helix bundle protein domain. This is understoodto mean that the sequence of the BM is “inserted” into or “grafted” ontothe sequence of the original three-helix bundle domain, such that the BMreplaces a similar structural motif in the original domain. For example,without wishing to be bound by theory, the BM is thought to constitutetwo of the three helices of a three-helix bundle, and can thereforereplace such a two-helix motif within any three-helix bundle. As theskilled person will realize, the replacement of two helices of thethree-helix bundle domain by the two BM helices has to be performed soas not to affect the basic structure of the polypeptide. That is, theoverall folding of the Ca backbone of the polypeptide according to thisembodiment of the invention is substantially the same as that of thethree-helix bundle protein domain of which it forms a part, e.g. havingthe same elements of secondary structure in the same order etc. Thus, aBM according to the disclosure “forms part” of a three-helix bundledomain if the polypeptide according to this embodiment of the aspect hasthe same fold as the original domain, implying that the basic structuralproperties are shared, those properties e.g. resulting in similar CDspectra. The skilled person is aware of other parameters that arerelevant.

In particular embodiments, the FcRn binding motif (BM) thus forms partof a three-helix bundle protein domain. For example, the BM mayessentially constitute two alpha helices with an interconnecting loop,within said three-helix bundle protein domain. In particularembodiments, said three-helix bundle protein domain is selected fromdomains of bacterial receptor proteins. Non-limiting examples of suchdomains are the five different three-helical domains of Protein A fromStaphylococcus aureus, such as domain B, and derivatives thereof. Insome embodiments, the three-helical bundle protein domain is a variantof protein Z, which is derived from domain B of staphylococcal ProteinA.

In embodiments where the FcRn binding polypeptide of the invention formspart of a three-helix bundle protein domain, the FcRn bindingpolypeptide may comprise an amino acid sequence selected from:

iii) (SEQ ID NO: 1077)K-[BM]-DPSQS X_(a)X_(b)LLX_(c) EAKKL X_(d)X_(e)X_(f)Q;wherein

[BM] is an FcRn binding motif as defined herein, provided that X₂₉ is D;

X_(a) is selected from A and S,

X_(b) is selected from N and E;

X_(c) is selected from A, S and C;

X_(d) is selected from E, N and S,

X_(e) is selected from D, E and S,

X_(f) is selected from A and S,

and

-   -   iv) an amino acid sequence which has at least 93% identity to a        sequence defined by iii).

In embodiments where the FcRn binding polypeptide of the invention formspart of a three-helix bundle protein domain, the FcRn bindingpolypeptide may comprise an amino acid sequence selected from:

v) (SEQ ID NO: 1080)K-[BM]-QPEQS X_(a)X_(b)LLX_(c) EAKKL X_(d)X_(e)X_(f)Q;wherein

[BM] is an FcRn binding motif as defined herein, provided that X₂₉ is R;

X_(a) is selected from A and S,

X_(b) is selected from N and E;

X_(c) is selected from A, S and C;

X_(d) is selected from E, N and S,

X_(e) is selected from D, E and S,

X_(f) is selected from A and S, and

-   -   vi) an amino acid sequence which has at least 93% identity to a        sequence defined by v).

As discussed above, polypeptides comprising minor changes as compared tothe above amino acid sequences which do not largely affect the tertiarystructure and the function thereof are also within the scope of thepresent disclosure. Thus, in some embodiments, sequence iv) or sequencevi) has at least 95%, for example at least 97% identity to a sequencedefined by iii) and v), respectively.

In one embodiment, X_(a) in sequence iii) or v) is A. In an alternativeembodiment, X_(a) in sequence iii) or v) is S.

In one embodiment, X_(b) in sequence iii) or v) is N. In an alternativeembodiment, X_(b) in sequence iii) or v) is E.

In one embodiment, X_(c) in sequence iii) or v) is A. In an alternativeembodiment, X₆ in sequence iii) or v) is S. In yet another alternativeembodiment, X_(c) in sequence iii) or v) is C.

In one embodiment, X_(d) in sequence iii) or v) is E.

In one embodiment, X_(d) in sequence iii) or v) is N.

In one embodiment, X_(d) in sequence iii) or v) is S.

In one embodiment, X_(e) in sequence iii) or v) is D.

In one embodiment, X₆ in sequence iii) or v) is E.

In one embodiment, X_(e) in sequence iii) or v) is S.

In one embodiment, X_(d)X_(e) in sequence iii) or v) is selected fromEE, ES, SE and SS.

In one embodiment, X_(d)X_(e) in sequence iii) or v) is ES.

In one embodiment, X_(d)X_(e) in sequence iii) or v) is SE.

In one embodiment, X_(f) in sequence iii) or v) is A. In an alternativeembodiment, X_(f) in sequence iii) or v) is S.

In one embodiment, in sequence iii) or v), X_(a) is A; X_(b) is N; X_(c)is A and X_(f) is A.

In one embodiment, in sequence iii) or v), X_(a) is A; X_(b) is N; X_(c)is C and X_(f) is A.

In one embodiment, in sequence iii) or v), X_(a) is S, X_(b) is E; X_(c)is S and X_(f) is S.

In one embodiment, in sequence iii) or v), X_(a) is S, X_(b) is E; X_(c)is C and X_(f) is S.

In one embodiment, in sequence iii) or v), X_(a) is A; X_(b) is N; X_(c)is A; X_(d)X_(e) is ND and X_(f) is A.

In one embodiment, in sequence iii) or v), X_(a) is A; X_(b) is N; X_(c)is C; X_(d)X_(e) is ND and X_(f) is A.

In one embodiment, in sequence iii) or v), X_(a) is S, X_(b) is E; X_(c)is S, X_(d)X_(e) is ND and X_(f) is S.

In one embodiment, in sequence iii) or v), X_(a) is S, X_(b) is E; X_(c)is C; X_(d)X_(e) is ND and X_(f) is S.

In one embodiment, in sequence iii) or v), X_(a) is A; X_(b) is N; X_(c)is A; X_(d)X_(e) is SE and X_(f) is A.

In one embodiment, in sequence iii) or v), X_(a) is A; X_(b) is N; X_(c)is C; X_(d)X_(e) is SE and X_(f) is A.

In one embodiment, in sequence iii) or v), X_(a) is S, X_(b) is E; X_(c)is S, X_(d)X_(e) is SE and X_(f) is S.

In one embodiment, in sequence iii) or v), X_(a) is S, X_(b) is E; X_(c)is C; X_(d)X_(e) is SE and X_(f) is S.

In one embodiment, in sequence iii) or v), X_(a) is A; X_(b) is N; X_(c)is A; X_(d)X_(e) is ES and X_(f) is A.

In one embodiment, in sequence iii) or v), X_(a) is A; X_(b) is N; X_(c)is C; X_(d)X_(e) is ES and X_(f) is A.

In one embodiment, in sequence iii) or v), X_(a) is S, X_(b) is E; X_(c)is S, X_(d)X_(e) is ES and X_(f) is S.

In one embodiment, in sequence iii) or v), X_(a) is S, X_(b) is E; X_(c)is C; X_(d)X_(e) is ES and X_(f) is S.

In yet a further embodiment, sequence iii) in the definition of FcRnbinding polypeptides above is selected from the group consisting of SEQID NO:354-706. In one embodiment, sequence iii) is selected from thegroup consisting of SEQ ID NO:354-368, SEQ ID NO:370-493 and SEQ IDNO:706. In one embodiment, sequence iii) is selected from the groupconsisting of SEQ ID NO:354-355 and SEQ ID NO:370-493. In oneembodiment, sequence iii) is selected from the group consisting of SEQID NO:354-355, SEQ ID NO:370-445, SEQ ID NO:447-456, SEQ ID NO:458-478and SEQ ID NO:480-493. In one embodiment, sequence iii) is selected fromthe group consisting of SEQ ID NO:354-361, SEQ ID NO:366, SEQ IDNO:372-373, SEQ ID NO:376, SEQ ID NO:381, SEQ ID NO:394, SEQ ID NO:397,SEQ ID NO:418, SEQ ID NO:423, SEQ ID NO:426, SEQ ID NO:428-430 and SEQID NO:706. In another embodiment, sequence iii) is selected from thegroup consisting of SEQ ID NO:354, SEQ ID NO:376, SEQ ID NO:381, SEQ IDNO:394, SEQ ID NO:397, SEQ ID NO:418, SEQ ID NO:426 and SEQ IDNO:428-430. In yet another embodiment, sequence iii) is selected fromSEQ ID NO:354, SEQ ID NO:376, SEQ ID NO:397, SEQ ID NO:418, SEQ IDNO:428 and SEQ ID NO:430. In one embodiment, sequence iii) is selectedfrom SEQ ID NO:354, SEQ ID NO:376 and SEQ ID NO:428. In one embodiment,sequence iii) is SEQ ID NO:354.

Also, in a further embodiment, there is provided an FcRn bindingpolypeptide as defined above, which comprises an amino acid sequenceselected from:

vii) (SEQ ID NO: 1081) YAK-[BM]-DPSQS SELLX_(c) EAKKL NDSQA P;wherein [BM] is an FcRn binding motif as defined above and X_(c) isselected from A, S and C; and

-   -   viii) an amino acid sequence which has at least 94% identity to        a sequence defined by vii).

Alternatively, there is provided an FcRn binding polypeptide as definedabove, which comprises an amino acid sequence selected from:

ix) (SEQ ID NO: 1082) FNK-[BM]-DPSQS ANLLX_(c) EAKKL NDAQA P;wherein [BM] is an FcRn binding motif as defined above and X_(c) isselected from A and C; and

-   -   x) an amino acid sequence which has at least 94% identity to a        sequence defined by ix).

As discussed above, polypeptides comprising minor changes as compared tothe above amino acid sequences that do not largely affect the tertiarystructure and the function thereof are also within the scope of thepresent disclosure. Thus, in some embodiments, the FcRn bindingpolypeptide as defined above may comprise a sequence which is at least96%, such as at least 98% identical to a sequence defined by vii) orix).

In some embodiments, the FcRn binding motif may form part of apolypeptide comprising an amino acid sequence selected from

(SEQ ID NO: 1083) ADNNFNK-[BM]-DPSQSANLLSEAKKLNESQAPK; (SEQ ID NO: 1084)ADNKFNK-[BM]-DPSQSANLLAEAKKLNDAQAPK; (SEQ ID NO: 1085)ADNKFNK-[BM]-DPSVSKEILAEAKKLNDAQAPK; (SEQ ID NO: 1086)ADAQQNNFNK-[BM]-DPSQSTNVLGEAKKLNESQAPK; (SEQ ID NO: 1087)AQHDE-[BM]-DPSQSANVLGEAQKLNDSQAPK; (SEQ ID NO: 1088)VDNKFNK-[BM]-DPSQSANLLAEAKKLNDAQAPK; (SEQ ID NO: 1089)AEAKYAK-[BM]-DPSESSELLSEAKKLNKSQAPK; (SEQ ID NO: 1090)VDAKYAK-[BM]-DPSQSSELLAEAKKLNDAQAPK; (SEQ ID NO: 1091)VDAKYAK-[BM]-DPSQSSELLAEAKKLNDSQAPK; (SEQ ID NO: 1092)AEAKYAK-[BM]-DPSQSSELLSEAKKLNDSQAPK; (SEQ ID NO: 1093)AEAKYAK-[BM]-DPSQSSELLSEAKKLSESQAPK; (SEQ ID NO: 1094)AEAKYAK-[BM]-DPSQSSELLSEAKKLESSQAPK; (SEQ ID NO: 1095)VDAKYAK-[BM]-DPSQSSELLSEAKKLNDSQAPK; (SEQ ID NO: 1096)VDAKYAK-[BM]-DPSQSSELLSEAKKLSESQAPK; (SEQ ID NO: 1097)VDAKYAK-[BM]-DPSQSSELLSEAKKLESSQAPK; (SEQ ID NO: 1098)VDAKYAK-[BM]-DPSQSSELLAEAKKLNKAQAPK; and (SEQ ID NO: 1099)AEAKYAK-[BM]-DPSQSSELLAEAKKLNKAQAPK;wherein [BM] is an FcRn binding motif as defined above.

In one embodiment, the FcRn binding polypeptide comprises an amino acidsequence selected from:

xi) (SEQ ID NO: 1078) AEAKYAK-[BM]-DPSQSSELLSEAKKLNDSQAPK;wherein [BM] is an FcRn binding motif as defined above; and

-   -   xii) an amino acid sequence which has at least 94% identity to        the sequence defined in xi).

In one embodiment, sequence xi) is selected from the group consisting ofSEQ ID NO:1060-1062.

In one embodiment, the FcRn binding polypeptide comprises an amino acidsequence selected from:

xiii) (SEQ ID NO: 1079) VDAKYAK-[BM]-DPSQSSELLSEAKKLNDSQAPK;wherein [BM] is an FcRn binding motif as defined above; and

-   -   xiv) an amino acid sequence which has at least 94% identity to        the sequence defined in xiii).

Sequence xiii) in such a polypeptide may for example be selected fromthe group consisting of SEQ ID NO:707-1059. In one embodiment, sequencexiii) is selected from the group consisting of SEQ ID NO:707-721, SEQ IDNO:723-846 and SEQ ID NO:1059. In one embodiment, sequence xiii) isselected from the group consisting of SEQ ID NO:707-708 and SEQ IDNO:723-846. In one embodiment, sequence xiii) is selected from the groupconsisting of SEQ ID NO:707-708, SEQ ID NO:723-798, SEQ ID NO:800-809,SEQ ID NO:811-831 and SEQ ID NO:833-846. In one embodiment, sequencexiii) is selected from the group consisting of SEQ ID NO:707-714, SEQ IDNO:719, SEQ ID NO:725-726, SEQ ID NO:729, SEQ ID NO:734, SEQ ID NO:747,SEQ ID NO:750, SEQ ID NO:771, SEQ ID NO:776, SEQ ID NO:779, SEQ IDNO:781-783 and SEQ ID NO:1059. In another embodiment, sequence xiii) isselected from the group consisting of SEQ ID NO:707, SEQ ID NO:729, SEQID NO:734, SEQ ID NO:747, SEQ ID NO:750, SEQ ID NO:771, SEQ ID NO:779and SEQ ID NO:781-783. In yet another embodiment, sequence xiii) isselected from SEQ ID NO:707, SEQ ID NO:729, SEQ ID NO:750, SEQ IDNO:771, SEQ ID NO:781 and SEQ ID NO:783. In one embodiment, sequencexiii) is selected from SEQ ID NO:707, SEQ ID NO:729 and SEQ ID NO:781.In one embodiment, sequence xiii) is SEQ ID NO:707.

Again, polypeptides comprising minor changes as compared to the aboveamino acid sequences which do not largely affect the tertiary structureand the function thereof are also within the scope of the presentdisclosure. Thus, in some embodiments, the FcRn binding polypeptide asdefined above may comprise a sequence which is at least 96%, such as atleast 98% identical to a sequence defined by xi) or xiii).

The terms “FcRn binding” and “binding affinity for FcRn” as used in thisspecification refer to a property of a polypeptide which may be testedfor example by the use of surface plasmon resonance (SPR) technology orELISA.

For example as described in the examples below, FcRn binding affinitymay be tested in an experiment in which FcRn, or a correctly foldedfragment thereof, is immobilized on a sensor chip of the instrument, andthe sample containing the polypeptide to be tested is passed over thechip. Alternatively, the polypeptide to be tested is immobilized on asensor chip of the instrument, and a sample containing FcRn, or acorrectly folded fragment thereof, is passed over the chip. The skilledperson may then interpret the results obtained by such experiments toestablish at least a qualitative measure of the binding affinity of thepolypeptide for FcRn. If a quantitative measure is desired, for exampleto determine a K_(D) value for the interaction, surface plasmonresonance methods may also be used. Binding values may for example bedefined in a Biacore (GE Healthcare) or ProteOn XPR 36 (Bio-Rad)instrument. FcRn is suitably immobilized on a sensor chip of theinstrument, and samples of the polypeptide whose affinity is to bedetermined are prepared by serial dilution and injected in random order.K_(D) values may then be calculated from the results using for examplethe 1:1 Langmuir binding model of the BIAevaluation 4.1 software, orother suitable software, provided by the instrument manufacturer.

Alternatively, as described in the examples below, FcRn binding affinitymay be tested in an experiment in which samples of the polypeptide arecaptured on antibody coated ELISA plates, and biotinylated FcRn is addedfollowed by streptavidin conjugated HRP. TMB substrate is added and theabsorbance at 450 nm is measured using a multi-well plate reader, suchas Victor³ (Perkin Elmer). The skilled person may then interpret theresults obtained by such experiments to establish at least a qualitativemeasure of the binding affinity of the polypeptide for FcRn. If aquantitative measure is desired, for example to determine the K_(D)value (the half maximal effective concentration) for the interaction,ELISA may also be used. The response of the polypeptides against adilution series of biotinylated FcRn are measured using ELISA asdescribed above. The skilled person may then interpret the resultsobtained by such experiments and K_(D) values may be calculated from theresults using for example GraphPad Prism 5 and non-linear regression.

In one embodiment, there is provided an FcRn binding polypeptide, whichis capable of binding to FcRn at pH 6.0 such that the K_(D) value of theinteraction is at most 1×10⁻⁶ M, such as at most 1×10⁻⁷ M, such as atmost 1×10⁻⁸ M, such as at most 1×10⁻⁹ M, such as at most 1×10⁻¹⁰ M. AnFcRn binding polypeptide according to this embodiment would bind, orremain bound, to FcRn in acidic pH conditions, such as pH 6.0, forexample in a lysosome. If such a polypeptide were to enter anincreasingly acidic intracellular environment, it would be recycled tothe plasma membrane through its interaction with FcRn, and thus avoiddegradation.

In one embodiment, the K_(D) value of the interaction between FcRnbinding polypeptide and FcRn at pH 7.4 is higher than the K_(D) value ofsaid interaction at pH 6.0. Thus, the FcRn binding polypeptide wouldbind to FcRn with higher affinity at pH 6.0 than at pH 7.4. In oneembodiment, the K_(D) value of said interaction at pH 7.4 is at least 2times higher, such as at least 5 times higher, such as at least 10 timeshigher, such as at least 50 times higher, such as at least 100 timeshigher than the K_(D) value of said interaction at pH 6.0.

In one embodiment, the K_(D) value of the interaction between FcRnbinding polypeptide and FcRn at pH 7.4 is at least 1×10⁻⁸ M, such as atleast 1×10⁻⁷ M, such as at least 1×10⁻⁶ M, such as at least 1×10⁻⁵ M. Insome embodiments, the only criterion for the interaction between FcRnbinding polypeptide and FcRn at pH 7.4 is that any FcRn bindingpolypeptide which has bound to FcRn during more acidic conditions isreleased more rapidly from FcRn when the pH value increases.

In an alternative embodiment, there is provided an FcRn bindingpolypeptide, for which the K_(D) of said interaction at pH 7.4 is thesame as or lower than the K_(D) of said interaction at pH 6.0. An FcRnbinding polypeptide according to this embodiment would bind or remainbound to FcRn in acidic pH conditions (i.e. would have an off-rate at pH6.0 which is sufficiently slow to avoid release), for example in thelysosome, as well as in neutral or slightly basic pH conditions, forexample on the plasma membrane. In a more specific embodiment, the K_(D)value of said interaction at pH 7.4 is at least 2 times lower, such asat least 5 times lower, such as at least 10 times lower, such as atleast 50 times lower, such as at least 100 times lower than the K_(D)value of said interaction at pH 6.0.

In another embodiment, there is provided an FcRn binding polypeptide,which is capable of binding to FcRn at pH 7.4 such that the K_(D) valueof the interaction is at most 1×10⁻⁶ M, such as at most 1×10⁻⁷ M, suchas at most 1×10⁻⁸ M, such as at most 1×10⁻⁹ M, such as at most 1×10⁻¹⁰M. An FcRn binding polypeptide according to this embodiment would bindor remain bound for an extended time to FcRn in neutral or slightlybasic pH conditions, such as pH 7.4, for example on the plasma membrane.The term “remain bound” should be understood to mean an interactionhaving a slow off-rate at given conditions.

In general, the skilled person knows that the K_(D) value of aninteraction is defined as the ratio between the off-rate (k_(off)) andthe on-rate (k_(on)). Thus, a high K_(D) value may be due to either ahigh k_(off), a low k_(on) or both, and conversely, a low K_(D) valuemay be due to either a low k_(off), a high k_(on) or both.

The skilled person will understand that various modifications and/oradditions can be made to an FcRn binding polypeptide according to anyaspect disclosed herein in order to tailor the polypeptide to a specificapplication without departing from the scope of the present disclosure.

For example, in one embodiment there is provided an FcRn bindingpolypeptide as described herein, which polypeptide has been extended byone or more amino acids at the C terminal and/or N terminal end. Such apolypeptide should be understood as a polypeptide having one or moreadditional amino acid residues at the very first and/or the very lastposition in the polypeptide chain. Thus, an FcRn binding polypeptide maycomprise any suitable number of additional amino acid residues, forexample at least one additional amino acid residue. Each additionalamino acid residue may individually or collectively be added in orderto, for example, improve production, purification, stabilization in vivoor in vitro, coupling, or detection of the polypeptide. Such additionalamino acid residues may comprise one or more amino acid residues addedfor the purpose of chemical coupling. One example of this is theaddition of a cysteine residue. Such additional amino acid residues mayalso provide a “tag” for purification or detection of the polypeptide,such as a His₆ tag or a “myc” (c-myc) tag or a “FLAG” tag forinteraction with antibodies specific to the tag or immobilized metalaffinity chromatography (IMAC) in the case of the hexahistidine tag.

The further amino acids as discussed above may be coupled to the FcRnbinding polypeptide by means of chemical conjugation (using knownorganic chemistry methods) or by any other means, such as expression ofthe FcRn binding polypeptide as a fusion protein or joined in any otherfashion, either directly or via a linker, for example an amino acidlinker.

The further amino acids as discussed above may for example comprise oneor more polypeptide domain(s). A further polypeptide domain may providethe FcRn binding polypeptide with another function, such as for exampleanother binding function, or an enzymatic function, or a toxic functionor a fluorescent signaling function, or combinations thereof.

A further polypeptide domain may moreover provide another FcRn bindingmoiety with the same FcRn binding function. Thus, in a furtherembodiment, there is provided an FcRn binding polypeptide in amultimeric form. Said multimer is understood to comprise at least twoFcRn binding polypeptides as disclosed herein as monomer units, theamino acid sequences of which may be the same or different. Multimericforms of the polypeptides may comprise a suitable number of domains,each having an FcRn binding motif, and each forming a monomer within themultimer. These domains may have the same amino acid sequence, butalternatively, they may have different amino acid sequences. In otherwords, the FcRn binding polypeptide of the invention may form homo- orheteromultimers, for example homo- or heterodimers. In one embodiment,there is provided an FcRn binding polypeptide, wherein said monomericunits are covalently coupled together. In another embodiment, said FcRnbinding polypeptide monomer units are expressed as a fusion protein. Inone embodiment, there is provided an FcRn binding polypeptide in dimericform.

Additionally, “heterogenic” fusion polypeptides or proteins, orconjugates, in which an FcRn binding polypeptide described herein, ormultimer thereof, constitutes a first domain, or first moiety, and thesecond and further moieties have other functions than binding FcRn, arealso contemplated and fall within the ambit of the present disclosure.The second and further moiety/moieties of the fusion polypeptide orconjugate in such a protein suitably have a desired biological activity.

Thus, in a second aspect of the present disclosure, there is provided afusion protein or a conjugate, comprising a first moiety consisting ofan FcRn binding polypeptide according to the first aspect, and a secondmoiety consisting of a polypeptide having a desired biological activity.In another embodiment, said fusion protein or conjugate may additionallycomprise further moieties, comprising desired biological activities thatcan be either the same or different from the biological activity of thesecond moiety.

Such heterogenic fusion polypeptides could also be used to createheteromultimeric complexes of higher order. One example is aheterodimeric complex of two fusion polypeptides, each comprising anFcRn binding polypeptide according to the present disclosure in fusionwith another moiety. Such a complex could for example form a heterodimerin vivo or in vitro and be held together by non-covalent and/or covalentinteractions. A specific example of such a complex is a Fab fragment, inwhich both the light chain and heavy chain are produced in fusion withone FcRn binding polypeptide each, and which may include an inter-domaindisulphide bond. Many biologically relevant, heterodimeric complexesknown to the skilled person may be constructed using FcRn binding fusionproteins as monomer units.

In one embodiment of said fusion protein or conjugate, the total size ofthe molecule is below the threshold for efficient renal clearance uponadministration to a mammalian subject.

In another embodiment of said fusion protein or conjugate, the totalsize of the molecule is above the threshold for efficient renalclearance upon administration to a mammalian subject.

In one embodiment, there is provided a fusion protein or conjugate,wherein the in vivo half-life of said fusion protein or conjugate islonger than the in vivo half-life of the polypeptide having the desiredbiological activity per se.

Non-limiting examples of a desired biological activity comprise atherapeutic activity, a binding activity, and an enzymatic activity.

In one embodiment, said desired biological activity is a bindingactivity to a selected target.

One example of such a binding activity is a binding activity, whichincreases the in vivo half-life of a fusion protein or conjugate. Thisfusion protein or conjugate may comprise at least one further moiety. Inone particular embodiment, said target is albumin, binding to whichincreases the in vivo half-life of said fusion protein or conjugate. Inone embodiment, said albumin binding activity is provided by an albuminbinding domain (ABD) of streptococcal protein G or a derivative thereof.For example, said fusion protein or conjugate, comprising at least onefurther moiety, may comprise [FcRn binding polypeptide moiety]−[albuminbinding moiety]−[moiety with affinity for selected target]. It is to beunderstood that the three moieties in this example may be arranged inany order from the N- to the C-terminal of the polypeptide.

In one embodiment, when a complex between a target and the fusionprotein or conjugate as described herein is formed (or maintained) atacidic pH, such as pH 6.0, the target is rescued from elimination bylysosomal degradation. Thus, target half-life is extended. Half-lifeextension implies that the elimination rate of a target is lower wheninteracting with said fusion protein or conjugate than the eliminationrate of the target molecule in the absence of said fusion protein orconjugate. Furthermore, it is desirable in this embodiment that thebinding of target by the fusion protein or conjugate should notinterfere substantially with the function of the target.

On the other hand, when a complex between the target and the fusionprotein or conjugate as described herein is not maintained or not formedat acidic pH, the target is directed to the subcellular lysosomes whereit is degraded.

In one embodiment, there is provided a fusion protein or conjugate,wherein the rate of elimination of a selected, undesirable target fromthe subject is increased. Increased elimination of an undesirable targetimplies increased elimination rate of the target from the body of themulticellular organism, as compared to a “normal” elimination rate ofthe target molecule per se, i.e. without previous interaction with thefusion protein or conjugate.

In another embodiment, binding of a selected undesirable target couldinactivate the function of the target, thereby blocking its biologicalactivity in situations where this is desirable. Such biological activitymay for example be activation or blocking of receptors or an enzymaticor otherwise toxic or undesirable activity. Such undesirable target maybe an endogenous hormone, enzyme, cytokine, chemokine or a target havingsome other biological activity. By using an inactivating target binding,the biological activity is blocked until the target is delivered fordegradation and released at a low pH value, and the target bindingfusion protein is recycled to circulation. This recycling of the targetbinding fusion protein (via its FcRn binding moiety) enables it to“catalyze” the removal of more than one molecule of the selectedundesirable target.

Undesirable targets may for example be foreign proteins and compounds,or naturally expressed proteins that display elevated levels in plasmafollowing a medical condition and where a therapeutic effect may beachieved by elimination of said protein. The undesired target is notnecessarily evenly distributed in the plasma but may be concentrated incertain regions, for example around a tumor or at sites of inflammation.

Non-limiting examples of targets are targets selected from the groupconsisting of allergens, amyloids, antibodies, auto-antigens, bloodclotting factors, hormones, tumor cells, drug molecules, cytokines,chemokines, proteases, hypersensitivity mediators, proinflammatoryfactors, toxins such as bacterial toxins and snake venoms; pollutants,metals and anti-oxidants.

Under certain conditions, such as in certain cancer diseases, it isdesired to remove endogenous molecules, for example VEGF, PDGF, HGF andother growth stimulatory hormones. Such molecules could also be targetedby a binding function in said fusion protein or conjugate.

Under other conditions, such as in certain immunological diseases, itmay be desirable to remove endogenous molecules transiently, such asselected interleukines or TNF. Such molecules could also be targeted bya binding function in said fusion protein or conjugate.

In one embodiment, the second moiety having a desired biologicalactivity is a therapeutically active polypeptide. Non-limiting examplesof therapeutically active polypeptides are biomolecules, such asmolecules selected from the group consisting of enzymes, for examplealgasidase a and β, glucocerebrosidase, laronidase, arylsulphatase,aglucosidase-α, asparaginase, Factor VII, Factor VIII, Factor IX andFactor Xa, hormones and growth factors, for example growth hormone,transforming growth factor-β2, erythropoietin, insulin, insulin-likegrowth factor-1, myostatin, bone-derived growth factor and glucagon-likepeptide-1; chemokines, for example CCL17, CCL19, CCL20, CCL21, CCL22,CCL27, XCL1 and CXC3CL1, and cytokines, for example interleukin (IL)-2,IL-4, IL-7, IL-10, IL-12, IL-15, IL-18, IL-22, IL-27, interferon(IFN)-α, IFN-β, IFN-γ, tumor necrosis factor, granulocyte-colonystimulating factor (G-CSF), macrophage-CSF, andgranulocyte/macrophage-CSF.

As the skilled person understands, the FcRn binding polypeptideaccording to the first aspect may be useful in a fusion protein or as aconjugate partner to any other moiety. Therefore, the above lists oftherapeutically active polypeptides should not be construed as limitingin any way.

Other possibilities for the creation of fusion polypeptides orconjugates are also contemplated. Thus, an FcRn binding polypeptideaccording to the first aspect of the invention may be covalently coupledto a second or further moiety or moieties, which in addition to orinstead of target binding exhibit other functions. One example is afusion between one or more FcRn binding polypeptide(s) and anenzymatically active polypeptide serving as a reporter or effectormoiety.

With regard to the description above of fusion proteins or conjugatesincorporating an FcRn binding polypeptide according to the disclosure,it is to be noted that the designation of first, second and furthermoieties is made for clarity reasons to distinguish between FcRn bindingpolypeptide or polypeptides according to the disclosure on the one hand,and moieties exhibiting other functions on the other hand. Thesedesignations are not intended to refer to the actual order of thedifferent domains in the polypeptide chain of the fusion protein orconjugate. Thus, for example, said first moiety may without restrictionappear at the N-terminal end, in the middle, or at the C-terminal end ofthe fusion protein or conjugate.

In one embodiment, there is provided an FcRn binding polypeptide, fusionprotein or conjugate, which binds to FcRn such that binding of IgG toFcRn is at least partially inhibited. This inhibition may be due tobinding of the FcRn binding polypeptide, fusion protein or conjugate tothe same, or an at least partially overlapping, region of FcRn as IgG.Alternatively, the FcRn binding polypeptide, fusion protein or conjugatemay bind to a different region of FcRn than IgG but sterically hinderthe binding of IgG to FcRn. Thus, the rate of elimination or clearanceof IgG from the circulatory system would increase due to increasedlysosomal degradation of IgG, because the FcRn mediated recycling of IgGwould be wholly or partially unavailable due to the occupation of FcRnbinding sites by the FcRn binding polypeptide according to the presentdisclosure. In other words, administration of FcRn binding polypeptide,fusion protein or conjugate or composition according to the presentdisclosure will act to increase the catabolism of circulating IgGantibodies.

In one embodiment, the K_(D) value of the interaction between the FcRnbinding polypeptide, fusion protein or conjugate and FcRn is lower thanthe K_(D) of the interaction between IgG and FcRn. This relationship maybe true at both pH 6.0 and pH 7.4, or at pH 6.0 only.

The above aspects furthermore encompass polypeptides in which the FcRnbinding polypeptide according to the first aspect, or the FcRn bindingpolypeptide as comprised in a fusion protein or conjugate according tothe second aspect, further comprises a label, such as a label selectedfrom the group consisting of fluorescent dyes and metals, chromophoricdyes, chemiluminescent compounds and bioluminescent proteins, enzymes,radionuclides and particles. Such labels may for example be used fordetection of the polypeptide.

In other embodiments, the labeled FcRn binding polypeptide is present asa moiety in a fusion protein or conjugate also comprising a secondmoiety having a desired biological activity and/or comprising a bindingfunction as described above. The label may in some instances be coupledonly to the FcRn binding polypeptide, and in some instances both to theFcRn binding polypeptide and to the second moiety of the conjugate orfusion protein. Furthermore, it is also possible that the label may becoupled to a second moiety only and not to the FcRn binding moiety.Hence, in yet another embodiment there is provided an FcRn bindingpolypeptide comprising a second moiety, wherein said label is coupled tothe second moiety only.

When reference is made to a labeled polypeptide, this should beunderstood as a reference to all aspects of polypeptides as describedherein, including fusion proteins and conjugates comprising an FcRnbinding polypeptide and a second and optionally further moieties. Thus,a labeled polypeptide may contain only the FcRn binding polypeptide ande.g. a therapeutic radionuclide, which may be chelated or covalentlycoupled to the FcRn binding polypeptide, or contain the FcRn bindingpolypeptide, a therapeutic radionuclide and a second moiety such as asmall molecule having a desired biological activity, for exampleresulting in a therapeutic efficacy.

In embodiments where the FcRn binding polypeptide, fusion protein orconjugate is radiolabeled, such a radiolabeled polypeptide may comprisea radionuclide. A majority of radionuclides have a metallic nature, areused in the ionic form, and are typically incapable of forming stablecovalent bonds with elements presented in proteins and peptides. Forthis reason, labeling of proteins and peptides with radioactive metalsis performed with the use of chelators, i.e. multidentate ligands, whichform non-covalent compounds, called chelates, with the metal ions. In anembodiment of the FcRn binding polypeptide, fusion protein or conjugate,the incorporation of a radionuclide is enabled through the provision ofa chelating environment, through which the radionuclide may becoordinated, chelated or complexed to the polypeptide.

One example of a chelator is the polyaminopolycarboxylate type ofchelator. Two classes of such polyaminopolycarboxylate chelators can bedistinguished: macrocyclic and acyclic chelators.

In one embodiment, the FcRn binding polypeptide, fusion protein orconjugate comprises a chelating environment provided by apolyaminopolycarboxylate chelator conjugated to the FcRn bindingpolypeptide via a thiol group of a cysteine residue or an epsilon aminegroup of a lysine residue.

The most commonly used macrocyclic chelators for radioisotopes ofindium, gallium, yttrium, bismuth, radioactinides and radiolanthanidesare different derivatives of DOTA(1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid). In oneembodiment, a chelating environment of the FcRn binding polypeptide,fusion protein or conjugate is provided by DOTA or a derivative thereof.More specifically, in one embodiment, the chelating polypeptidesencompassed by the present disclosure are obtained by reacting the DOTAderivative 1,4,7,10-tetraazacyclododecane-1,4,7-tris-aceticacid-10-maleimidoethylacetamide (maleimidomonoamide-DOTA) with saidpolypeptide.

Additionally, 1,4,7-triazacyclononane-1,4,7-triacetic acid (NOTA) andderivatives thereof may be used as chelators. Hence, in one embodiment,there is provided an FcRn binding polypeptide, fusion protein orconjugate, wherein the polyaminopolycarboxylate chelator is1,4,7-triazacyclononane-1,4,7-triacetic acid or a derivative thereof.

The most commonly used acyclic polyaminopolycarboxylate chelators aredifferent derivatives of DTPA (diethylenetriamine-pentaacetic acid).Hence, polypeptides having a chelating environment provided bydiethylenetriaminepentaacetic acid or derivatives thereof are alsoencompassed by the present disclosure.

In a further embodiment, the FcRn binding polypeptide, producedrecombinantly through expression of a polynucleotide or synthetically,is conjugated to one or more synthetic polymers, in order for example toincrease its hydrodynamic radius. Polyethylene glycol (PEG) is commonlyused for this purpose, but other polymers have also been used in theart. Such “PEGylation” may be used to increase the size of the FcRnbinding polypeptide of any of the types described herein to a size abovethe threshold for effective renal excretion.

In one embodiment, a synthetic polymer is conjugated to one or morechemically synthesized, monomeric FcRn binding polypeptides. Otherfunctionalities may also be conjugated to the same synthetic polymer. Ifthe FcRn binding polypeptide and other components are chemicallysynthesized, none of the components will have to be made in a biologicalsystem if this is not desired.

In a preferred embodiment, one or more synthetically or biologicallymanufactured FcRn binding polypeptides are conjugated to a syntheticpolymer, to achieve a size exceeding the size associated with efficientrenal clearance and used for blocking binding of IgG to FcRn. A uniquecysteine in each FcRn binding polypeptide may be used for site specificconjugation, for example a C-terminally located cysteine introduced forthis purpose. With a branched synthetic polymer, more than two FcRnbinding moieties may be conjugated to the same polymer, to enhance theavidity and therefore the blocking potency.

In a third aspect of the present disclosure, there is provided apolynucleotide encoding an FcRn binding polypeptide or a fusion proteinas described herein. Also encompassed by this disclosure is a method ofproducing a polypeptide or fusion protein as described above comprisingexpressing a polynucleotide; an expression vector comprising thepolynucleotide; and a host cell comprising the expression vector.

Also encompassed is a method of producing a polypeptide, comprisingculturing said host cell under conditions permissive of expression ofsaid polypeptide from its expression vector, and isolating thepolypeptide.

The FcRn binding polypeptide of the present disclosure may alternativelybe produced by non-biological peptide synthesis using amino acids and/oramino acid derivatives having protected reactive side-chains, thenon-biological peptide synthesis comprising

-   -   step-wise coupling of the amino acids and/or the amino acid        derivatives to form a polypeptide according to the first aspect        having protected reactive side-chains,    -   removal of the protecting groups from the reactive side-chains        of the polypeptide, and    -   folding of the polypeptide in aqueous solution.

In a fourth aspect of the disclosure, there is provided a compositioncomprising an FcRn binding polypeptide, fusion protein or conjugate asdescribed herein and at least one pharmaceutically acceptable excipientor carrier. In one embodiment thereof, said composition furthercomprises at least one additional active agent, such as at least twoadditional active agents, such as at least three additional activeagents. Non-limiting examples of additional active agents that may proveuseful in such a combination are immunosuppressing agents,anti-inflammatory agents, anti-microbial agents and enzymes.

In one embodiment of this aspect, said composition is adapted foradministration by a route selected from the group consisting of oraladministration, intranasal administration, pulmonar administration,vaginal administration, rectal administration, intravenous injection,intraperitoneal injection, intramuscular injection, subcutaneousinjection and intradermal injection.

As used herein, the term “systemic administration” refers to a route ofadministration such the substance of interest enters into thecirculatory system so that the entire body is affected. The skilledperson is aware that systemic administration can take place via enteraladministration (absorption of the drug through the gastrointestinaltract) or parenteral administration (generally injection, infusion orimplantation).

In one embodiment, said composition is adapted for administrationsystemically or locally. In certain embodiments, systemic administrationof said compound may be used. In another embodiment, said composition isadapted for administration by a local route. For example, localadministration may be topical in an ointment, paste, foam or cream. Inanother embodiment, said composition is adapted for administrationacross an endothelial or epithelial layer. Here, the composition may betranscytosed across said layer.

In one embodiment, the rate of uptake of a composition comprising afusion protein or conjugate as described herein is higher than the rateof uptake of polypeptides corresponding to second or further moietiesper se. In one embodiment, the rate of uptake is at least 2 timeshigher, such as at least 5 times higher, such as at least 10 timeshigher, such as at least 25 times higher than the rate of uptake of theat second or further moieties per se.

It should be understood from the above disclosure that the FcRn bindingpolypeptide fusion protein or conjugate or the composition as describedherein may for example be useful as a therapeutic agent, and/or as ameans for extending the in vivo half-life of a fusion partner, and/or asa means for increasing the rate of elimination of undesirable targets.

Hence, in a fifth aspect of the present disclosure, there is provided anFcRn binding polypeptide, fusion protein, conjugate or composition asdisclosed herein for use as a medicament.

In a related, sixth, aspect of the present disclosure, there is provideda method of treatment of a subject in need thereof, comprising the stepof administrating a therapeutically active amount of an FcRn bindingpolypeptide, fusion protein, conjugate or composition as disclosedherein.

In one embodiment of any one of these two latter aspects, the medicamentor method is intended for treatment in which the capacity of the FcRnbinding polypeptide to at least partially block binding of IgG to FcRnis exploited, i.e. treatment in which increased catabolism of IgGantibodies is desired. In one embodiment, a condition in which suchtreatement may be indicated is an auto-immune condition. As non-limitingexamples of indicated conditions, mention is made of myasthenia gravis,Guillain-Barré syndrome, autoimmune limbic encephalitis, pediatricautoimmune neuropsychiatric disorders associated with streptococcalinfection (PANDAS), neuromyotonia (Isaac's syndrome), morvan syndrome,multiple sclerosis, pemphigus vulgaris, foliaceus, bullous pemphigoid,epidermolysis bullosa acquisita, pemphigoid gestationis, mucous membranepemphigoid, lichen sclerosus, antiphospholipid syndrome, erlapsingpolychondritis, autoimmune anemia, idiopathic trombocytic purpura,autoimmune Grave's disease, dilated cardiomyopathy, vasculitis,Goodpasture's syndrome, idiopathic membranous nephropathy, rheumatoidarthritis and systemic lupus erythematosus.

In another embodiment, there is provided an FcRn binding polypeptide,fusion protein, conjugate or composition as described herein for use inblocking or removal of an undesirable target from the circulation. Inone embodiment, said undesirable target is selected from the groupcomprising allergens, amyloids, antibodies, auto-antigens, bloodclotting factors, hormones, tumor cells, drug molecules, cytokines,chemokines, hypersensitivity mediators, pro-inflammatory factors, toxinssuch as bacterial toxins and snake venoms, pollutants, metals andanti-oxidants.

While the invention has been described with reference to variousexemplary aspects and embodiments, it will be understood by thoseskilled in the art that various changes may be made and equivalents maybe substituted for elements thereof without departing from the scope ofthe invention. In addition, many modifications may be made to adapt aparticular situation or molecule to the teachings of the inventionwithout departing from the essential scope thereof. Therefore, it isintended that the invention not be limited to any particular embodimentcontemplated, but that the invention will include all embodimentsfalling within the scope of the appended claims.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1A-1MM is a listing of the amino acid sequences of examples of FcRnbinding motifs comprised in FcRn binding polypeptides of the invention(SEQ ID NO:1-353), examples of 49-mer FcRn binding polypeptidesaccording to the disclosure (SEQ ID NO:354-706), examples of 58-mer FcRnbinding polypeptides according to the disclosure (SEQ ID NO:707-1062) aswell as the amino acid sequences of the albumin binding polypeptidevariant PP013 (SEQ ID NO:1063), Taq polymerase binding Z variant Z03638(SEQ ID NO:1064), human αFcRn (SEQ ID NO:1065), murine αFcRn (SEQ IDNO:1070), human δ2-microglobulin (SEQ ID NO:1066), murineβ2-microglobulin (SEQ ID NO:1067), human αFcRn (SEQ ID NO:1068) when inhuman FcRn-eGFP and murine αFcRn (SEQ ID NO:1069) when in murineFcRn-eGFP.

FIGS. 2A-2E show the binding to human FcRn at pH 6.0 and dissociationsat pH 6.0 and 7.4 for His₆-tagged Z variants and for IgG as described inExample 3. Overlays of sensorgrams obtained from a Biacore instrumentrepresenting injection at pH 6.0 followed by dissociation at pH 6.0(solid line) and injection at pH 6.0 followed by dissociation at pH 7.4(dashed line) are displayed for (A) Z07918 (SEQ ID NO:707), (B) Z07960(SEQ ID NO:710), (C) Z10109 (SEQ ID NO:709), (D) Z10193 (SEQ ID NO:708)and (E) IgG.

FIG. 3 shows dot plots from a flow cytometry analysis of binding of FcRnbinding Z variant to human (upper panel) and mouse (lower panel)FcRn-eGFP HeLa cells, as described in Example 4. Due to heterogeneousexpression of FcRn-eGFP by HeLa cells, cells were gated according toFcRn-eGFP expression level. Cells in gate H are considered to beFcRn-eGFP negative and cells in gate I are considered to be positive.Incubation with Alexa647 labeled Z variants resulted in a populationpositive both for Alexa647 and eGFP, whereas incubation with buffer(buffer control) did not. The figure shows that the three variantsZ07960 (SEQ ID NO:710), Z07930 (SEQ ID NO:712) and Z07918 (SEQ IDNO:707) bind to human FcRn and mouse FcRn. The y-axis shows Alexa647intensity and the x-axis shows eGFP activity.

FIG. 4 shows mean fluorescence intensity (MFI) values of Alexa647labeled Z07960 (SEQ ID NO:710), Z07930 (SEQ ID NO:712) and Z07918 (SEQID NO:707), measured in the cell binding assay described in Example 4.Diagram (A) shows MFI from HeLa cells transduced with human FcRn-eGFPand diagram (B) shows MFI from HeLa cells transduced with mouseFcRn-eGFP.

FIG. 5 shows dot plots from flow cytometry analysis of human or mouseIgG Alexa647 binding to human (upper panel) and mouse (lower panel)FcRn-eGFP HeLa cells, as described in Example 5. Due to heterogeneousexpression of FcRn-eGFP by HeLa cells, cells were gated according to theabundance of FcRn-eGFP on the cell surface. Cells in gate M areconsidered to be FcRn-eGFP negative and cells in gate N are consideredto be positive. Binding of 100 nM human or mouse IgG-Alexa647 to FcRntransduced HeLa cells are shown in the left panel (0 nM). The figureshows that IgG binding was blocked by His₆-tagged Z07918 (SEQ ID NO:707)in a dose dependent manner (1, 10, 100 and 1000 nM). The y-axis showsAlexa647 intensity and the x-axis shows eGFP activity.

FIG. 6 shows mean fluorescence intensity (MFI) values resulting fromFcRn binding of IgG Alexa647 in the presence of different concentrationsof His₆-tagged Z07918 (SEQ ID NO:707) on (A) human FcRn-eGFP transducedHeLa cells and (B) mouse FcRn-eGFP transduced HeLa cells, as describedin Example 5. The figure shows dose dependent blocking of the IgG-FcRnbinding by the Z variant.

FIGS. 7A-7C show kinetics of binding of three Z variants to human FcRnat pH 6.0, as described in Example 6, using a Biacore instrument.Sensorgrams for a concentration series of (A) Z11948 (SEQ ID NO:1060),(B) Z11946 (SEQ ID NO:1061) and (C) Z11947 (SEQ ID NO:1062),respectively, in fusion with the albumin binding polypeptide PP013 (SEQID NO:1063) and the control Z variant molecule Z03638 (SEQ ID NO:1064,not specific for FcRn), are displayed. Curves from 640 nM (dashed line),160 nM (dotted line) and 40 nM (solid grey line) were subjected tokinetic analysis using the Langmuir 1:1 binding model. Kineticparameters and affinities were calculated from fitted curves (solidblack lines) and are shown in Table 5.

FIG. 8 shows the pharmacokinetic profiles for three FcRn binding Zvariants fused to the albumin binding polypeptide PP013 obtained asdescribed in Example 6. The Z variants Z11947 (SEQ ID NO: 1062, opensquares), Z11946 (SEQ ID NO:1061, open triangles) and Z11948 (SEQ IDNO:1060, open diamonds) all displayed prolonged half-life compared tothe negative control PP013-Z03638 (open circles).

FIG. 9 shows the blocking of human IgG to human FcRn by His₆-Z07918 (SEQID NO:707; black circles), IVIg (grey squares) and SCIg (greytriangles), respectively, assayed as described in Example 10.

FIG. 10 shows that blocking of the IgG-FcRn interactions with FcRnspecific Z variants in mice results in reduced levels of IgG. As furtherdescribed in Example 11, mice were treated with five daily injections ofVehicle (+), the ABD fused Z variant Z07918-PP013 (open square) andZ11948 (SEQ ID NO:1060, closed circle). The concentration of endogenousIgG was measured by ELISA. The concentration of IgG in individual miceat 24, 72, 120 and 168 h were related to the level at 0 h and theresults are therefore presented as percentage of IgG at 0 h.

EXAMPLES Summary

The following Examples disclose the development of novel Z variantmolecules targeting the neonatal Fc receptor (FcRn). The Z variants wereobtained using phage display technology. The genes encoding FcRn bindingpolypeptides described herein were sequenced, and the correspondingamino acid sequences are listed in FIG. 1A-1MM, and denoted by theidentifiers SEQ ID NO:707-1059. Also, the deduced binding motifs ofthese selected binding variants are listed in FIG. 1A-1MM with sequenceidentifiers SEQ ID NO:1-353.

Example 1 Production of Human αFcRn and Human β2-Microglobulin (B2M)

In this Example, the extracellular domain (ECD) of human αFcRn (SEQ IDNO:1065) in complex with human β2-microglobulin (SEQ ID NO:1066)(complex denoted FcRn) and human β2-microglobulin in non-complexed form(denoted B2M) were produced as soluble proteins. Human FcRn and B2Mproduced in this Example were used for phage selection, ELISA andBiacore assays in Examples 2 and 3.

Materials and Methods

Construction of Plasmids Containing the Genes for Human αFcRn and Humanβ2-Microglobulin to be Used for Co-Expression:

The genes encoding human αFcRn (Genbank BC008734.2) and humanβ2-microglobulin (B2M) (Genbank BC032589.1) were obtained fromOpenBiosystems. Using PCR overlap extension, a gene fragment encodingamino acids 24-290 of human αFcRn (αFcRn_(ECD)) (SEQ ID NO:1065) wasamplified to a construct consisting of attB1-site/Kozak sequencefollowed by a gene encoding: an Ig kappa chain leader sequence,hFcRn_(ECD), a GS-linker and a flag tag, followed by an attB2 site. Asimilar construct was made containing a gene fragment encoding aminoacids 21-119 of human B2M (SEQ ID NO:1066), except that a His₆ tagreplaced the flag tag. The constructs were inserted into the plasmidpDONOR221 (Invitrogen, cat. no. 12536-017) by recombination using theGateway system (Invitrogen, cat. no. 11789020, GATEWAY BP CLONASE IIEnzyme mix), according to the manufacturer's recommendations. Afterverification of correct sequences, the human αFcRn_(ECD) construct wasinserted into 2K7_(bsd) (Suter et al. (2006) Stem Cells 24:615-623)using multi-site gateway cloning together with the promoter-containingplasmid pENTR-CMV (Tai et al. (2012) PLoS One 7(9):e46269), resulting inthe vector 2K7_(bsd)-CMV-hFcRn_(ECD). The human B2M gene construct wassimilarly inserted into 2K7_(neo) (Suter et al., supra), giving thevector 2K7_(neo)-CMV-hB2M.

Cell Culture, Preparation of Recombinant Lentiviral Vectors and GeneInsertions into SKOV-3 Cell Line:

The HEK293T and SKOV-3 cell lines were obtained from ATCC. Cells weregrown at 37° C. in a humidified incubator in the presence of 5% CO₂.Complete medium for the HEK293T cell line was Dulbeccos modified eaglemedium (DMEM) supplemented with 10% fetal bovine serum (FBS), 1%Antibiotic Antimycotic Solution (AA) and 1% MEM Non-essential Amino AcidSolution (NEAA). Complete medium for the SKOV-3 cell line was McCoy's 5Amedium supplemented with 10% FBS and 1% AA.

The plasmids 2K7_(bsd)-CMV-hFcRn_(ECD) and 2K7_(neo)-CMV-hB2M wereseparately co-transfected together with VSV-G envelope and gag/polpackaging plasmid into HEK293T cells using calcium chloride transfection(Zufferey et al. (1997) Nat Biotechnol 15(9):871-5, Jakobsson et al.(2006) J Neurosci Res 84:58-67). HEK293 culture supernatants containingformed lentiviral particles with human αFcRn_(ECD) and human B2Mtransgenes, respectively, were cleared from cell debris bycentrifugation and filtration. The two types of lentiviral particleswere used to sequentially transduce SKOV-3 cells. Successful doubleintegrants containing both the human αFcRn_(ECD) and the B2M genes wereselected for by the addition of blasticidin (Invitrogen) and G418sulfate (Invitrogen) to culture medium while passaging the cells for twoweeks. The resulting, stably transduced SKOV-3 cell line was denotedSKOV-3 hFcRn_(ECD)/hB2M.

Expression of Recombinant Human FcRn:

SKOV-3 cells, co-expressing human αFcRn_(ECD) and B2M resulting in humanFcRn, were expanded and 1.5×10⁷ cells were seeded in a HYPERFIask(Corning) in 560 ml complete growth medium. After five days, when thecells had settled and multiplied, the medium was changed to completegrowth medium without FBS. After five days, the culture was terminatedand the supernatant was collected, passed through a 45 μm filter andfrozen at −80° C.

Purification of Recombinant Human FcRn Using Human IgG Chromatography:

Protein purification was carried out in an ÄKTA Explorer system (GEHealthcare). Human IgG (Pharmacia), 1 ml in 0.2 M NaHCO₃, 0.5 M NaCl pH8.3 at a concentration of 10 mg/ml, was coupled to a 1 ml HiTrapNHS-activated HP column (GE Healthcare) according to the manufacturer'sinstruction. The supernatant containing recombinant human FcRn fromSKOV-3 cells was thawed and the pH was adjusted to 5.8 with HCl. Thesupernatant was subsequently loaded in batches of 100 ml onto the columnpreviously equilibrated with 20 mM Bis-Tris pH 5.8. The column waswashed with 20 ml of 20 mM Bis-Tris pH 5.8 and eluted in fractions of 1ml using 50 mM Tris, pH 8.1. Buffer exchange to PBS (phosphate bufferedsaline, 10 mM phosphate, 137 mM NaCl, 2.68 mM KCl, pH 7.4) was performedusing dialysis.

SDS-PAGE and Western Blot:

The purity of the eluted fractions from the protein purification wasanalyzed by SDS-PAGE and staining with GelCode Blue Stain Reagent(Pierce) and SILVERXPRESS Silver Staining Kit (Invitrogen). Westernblotting was carried out using an Amersham HYBOND-C Extra nitrocellulosemembrane (GE Healthcare). The membrane was blocked with 5% non-fat drymilk (Semper) in TBS+T (50 mM Trizma base, 150 mM NaCl, 0.05% Tween-20,pH 8) for 1 hour, then probed with a mixture of rabbit anti-FCGRTpolyclonal antibody (Atlas Antibodies) at a concentration of 0.15 μg/mland rabbit anti-B2M polyclonal antibody (Atlas Antibodies) at aconcentration of 0.23 μg/ml in TBS+T. The membrane was subsequentlyincubated with stabilized goat anti-rabbit antibody conjugated withhorse radish peroxidase (Pierce) diluted 1:10,000 in TBS+T. Afteraddition of TMB Substrate (Pierce), an image of the membrane wasacquired on Amersham Hyperfilm ECL (GE Healthcare). The Hyperfilm wasprocessed using GBX developer and GBX fixer (Sigma-Aldrich).

Production of a Non-Complexed Form of Human B2M:

Human B2M was produced in E. coli. The expression and purification wasperformed essentially as described in Sandalova et al. (2005) ActaChryst F61:1090-1093 and Michaelsson et al. (2001) J Immunol166:7327-7334. The purified protein, consisting of amino acids 21-119 ofhuman B2M, in urea was subjected to arginine refolding as follows; 0.5mg of B2M was rapidly added to 2 ml refolding buffer (20 ml 1 M Tris-HClpH 8.0, 16.87 g L-Arginine (buffered with HCl), 0.8 ml 0.5 M EDTA, 61 mgGSSG, 307 mg GSH and milli-Q water to a final volume of 200 ml, pH 8.0,and supplemented with protease inhibitor (Roche, cat. no. 11 873 580001)). The refolding procedure was performed at 4° C. during 4 hours.Refolded B2M protein was buffer exchanged to PBS using a PD-10 column(GE Healthcare).

Results

Construction of Plasmids Containing the Genes for Human αFcRn and Humanβ2-Microglobulin to be Used for Co-Expression:

Genes encoding the extracellular domain of the α-chain of human FcRn(αFcRn_(ECD)) and human B2M were inserted into the lentiviral transferplasmids 2K7_(bsd) and 2K7_(neo), respectively. In both cases, theinserted gene is under the control of a CMV promoter. The genes wereextended so that the resulting proteins would have an Ig kappa chainleader sequence in the N-terminus to target the protein for exportthrough the endoplasmic reticulum to the culture medium (the signalsequence was cleaved upon secretion). In addition, αFcRn_(ECD) had aC-terminal spacer sequence followed by a FLAG-tag for potentialdetection. Human B2M had a C-terminal spacer sequence followed by a His₆tag for potential detection. The spacer sequence was added to enhanceaccessibility of the tag. The lentiviral transfer plasmids alsocontained two different antibiotic resistance genes to allow selectionof cells where both constructs had been inserted.

Expression and Purification of Recombinant Human FcRn:

The genes encoding αFcRn_(ECD) and B2M were inserted into the genome ofSKOV-3 by lentiviruses, and the resulting FcRn protein was secreted intothe culture medium. To capture only FcRn having retained pH-dependentIgG binding, affinity chromatography using immobilized IgG was usedwhere the receptor was captured at pH 5.8 and eluted at pH 8.1. Capturedprotein was eluted in three fractions.

SDS-PAGE and Western Blot:

To investigate the presence of two peptide chains (αFcRn_(ECD) and B2M)of the produced FcRn protein, and to analyze the purity of the elutedmaterial, an SDS-PAGE analysis was performed on the eluted fractions.For the gel stained with GelCode Blue Stain, two bands were detectedwith molecular weights of 12 and 36 kDa, respectively. This correspondsapproximately to the theoretical molecular weights of thenon-glycosylated peptide chains of 12 kDa for B2M and 31 kDa forαFcRn_(ECD). The αFcRn_(ECD) part of the protein contains oneglycosylation site and it was therefore expected that its molecular masswould be higher than 31 kDa. The gel was also silver stained to increasesensitivity and possibly detect impurities. A band of approximately 66kDa was detected in the first eluted fraction, which could correspond toBSA (bovine serum albumin) originating from cell attachment. The totalamount of protein recovered in fraction 2 and 3 corresponded to 1.4 mg/lculture medium. A western blot analysis on the pooled material wascarried out, which showed essentially only the two major bands and inaddition a very weak band below 12 kDa which might correspond to adegradation product.

Example 2 Selection and ELISA Binding of FcRn Binding Z Variants

In this Example, human FcRn was used as target in phage displayselections using a phage library of Z variants. Selected clones were DNAsequenced, produced in E. coli periplasmic fractions and assayed againstFcRn in ELISA (enzyme-linked immunosorbent assay).

Materials and Methods

Biotinylation of Target Protein FcRn and of B2M:

Human FcRn and human B2M, produced as described in Example 1, werebiotinylated using No-Weigh EZ-Link Sulfo-NHS-LC-Biotin (Pierce, cat.no. 21327) at a 31×(FcRn) and 10×(B2M) molar excess, respectively,according to the manufacturer's recommendations. The reactions wereperformed at room temperature (RT) for 30 min. Subsequent bufferexchange to PBS was performed using Slide-a-lyzer dialysis cassettes(FcRn; Pierce, cat. no. 66380, 10,000 MWCO and B2M; Pierce, cat. no.66333, 3,500 MWCO), according to the manufacturer's instructions.

Phage Display Selection of FcRn Binding Z Variants:

A library of random variants of protein Z displayed on bacteriophage,constructed in phagemid pAY02592 essentially as described in Grönwall etal. (2007) J Biotechnol, 128:162-183, was used to select FcRn binding Zvariants. In this library, an albumin binding domain (ABD, GA3 ofprotein G from Streptococcus strain G148) is used as fusion partner tothe Z variants. The library is denoted Zlib006Naive.II and has a size of1.5×10¹⁰ library members (Z variants). E. coli RRIΔM15 cells (Rüther etal., (1982) Nucleic Acids Res 10:5765-5772) from a glycerol stockcontaining the phagemid library Zlib006Naive.II, were inoculated in 20 lof a defined proline free medium [dipotassium hydrogenphosphate 7 g/l,trisodium citrate dihydrate 1 g/l, uracil 0.02 g/l, YNB (DIFCO YeastNitrogen Base w/o amino acids, Becton Dickinson) 6.7 g/l, glucosemonohydrate 5.5 g/l, L-alanine 0.3 g/l, L-arginine monohydrochloride0.24 g/l, L-asparagine monohydrate 0.11 g/l, L-cysteine 0.1 g/l,L-glutamic acid 0.3 g/l, L-glutamine 0.1 g/l, glycine 0.2 g/l,L-histidine 0.05 g/l, L-isoleucine 0.1 g/l, L-leucine 0.1 g/l, L-lysinemonohydrochloride 0.25 g/l, L-methionine 0.1 g/l, L-phenylalanine 0.2g/l, L-serine 0.3 g/l, L-threonine 0.2 g/I, L-tryptophane 0.1 g/l,L-tyrosine 0.05 g/l, L-valine 0.1 g/l supplemented with 100 μg/mlampicillin. The cultivations were grown at 37° C. in a fermenter (BelachBioteknik, BR20). When the cells reached an optical density at 600 nm(OD600) of 0.75, approximately 2.6 l of the cultivation was infectedusing a 10 × molar excess of M13K07 helper phage (New England Biolabs,cat. no. N0315S). The cells were incubated for 30 minutes, whereupon thefermenter was filled up to 20 l with TSB-YE (Tryptic Soy Broth-YeastExtract; 30 g/l TSB, 5 g/l yeast extract) supplemented with 100 μMisopropyl-β-D-1-thiogalactopyranoside (IPTG) for induction of expressionand with 25 μg/ml kanamycin and 12.5 μg/ml carbenicillin and grown at30° C. for 22 h. The cells in the cultivation were pelleted bycentrifugation at 15,900 g. The phage particles were precipitated fromthe supernatant twice in PEG/NaCl (polyethylene glycol/sodium chloride),filtered and dissolved in PBS and glycerol as described in Grönwall etal., supra. Phage stocks were stored at −80° C. before use.

Selections against biotinylated human FcRn were performed in four cyclesdivided in two different tracks. Phage stock preparation and selectionprocedure were performed essentially as described for selection againstanother biotinylated target in WO2009/077175. The amplification of phagebetween the selection cycles was performed by infecting E. coli RRIΔM15with phage, then performing cultivation in solution as follows. Elutedphage and 10× excess of M13K07 helper phage compared to bacteria wereallowed to simultaneously infect log phase bacteria at 37° C. for 30 minwithout rotation, followed by 30 min with slow rotation. Prior toinfection, bacteria were grown to log phase in the defined proline freemedium described above. Infected bacteria were pelleted bycentrifugation at 4,300 g for 10 min and resuspended in 200 ml TSB+YEmedium supplemented with 0.1 mM IPTG, 25 μg/ml kanamycin and 100 μg/mlampicillin and cultivated at 30° C. overnight for phage production.

The selection buffer consisted of 100 mM sodium phosphate and 150 mMsodium chloride adjusted to pH 5.5 with hydrogen chloride andsupplemented with 0.1% gelatin and 0.1% Tween-20. At selection, humanserum albumin (HSA, Albucult, Novozymes) was added to the selectionbuffer to a final concentration of 1.5 μM. In order to reduce the amountof background binders, pre-selection was performed by incubation ofphage stock with DYNABEADS M-280 Streptavidin (SA-beads, Dynal, cat. no.112.06) for 1 hour at RT. A second pre-selection was performed during 30min at RT against human B2M immobilized in immunotubes (Nunc, cat. no.444474). 5 μg/ml of human B2M in carbonate buffer (Sigma, cat. no.068K8214) was immobilized in the tube at 7° C. for >1 h. After washingtwice with tap water, the tubes were blocked with PBS+0.5% casein(Sigma, cat. no. C8654) for 30 min at RT before use. All tubes and beadsused in the selection were pre-blocked with PBS+0.1% gelatin. Selectionwas performed in solution at RT, followed by capture of target-phagecomplexes on SA-beads where 1 mg beads per 2.9 μg biotinylated FcRn wereused. In cycle 1 of the selections, 100 nM biotinylated FcRn was usedand two washes of two min each were performed using selection buffer. Anincreased stringency, using a lowered target concentration and anincreased number of washes, was applied in the subsequent cycles: 50nM/5 washes, 25 nM/8 washes and 10 nM/12 washes were applied in cycle 2,3 and 4, respectively. After the washes, bound phage was eluted from thetwo selection tracks using two different procedures; 1) 500 μl 0.1 Mglycine-HCl, pH 2.2, followed by immediate neutralization with 50 μl 1 MTris-HCl, pH 8.0, and 450 μl PBS, or; 2) 500 μl of 100 mM sodiumphosphate and 150 mM sodium chloride, pH 8.0 and neutralization with 500μl PBS.

Sequencing:

PCR fragments were amplified from single colonies using a standard PCRprogram and the primers AFFI-21 (5′-tgcttccggctcgtatgttgtgtg (SEQ IDNO:1071)) and AFFI-22 (5′-cggaaccagagccaccaccgg (SEQ ID NO:1072)).Sequencing of amplified fragments was performed using the biotinylatedoligonucleotide AFFI-72 (5′-biotin-cggaaccagagccaccaccgg (SEQ IDNO:1073)) and a BIGDYE Terminator v3.1 Cycle Sequencing Kit (AppliedBiosystems), used in accordance with the manufacturer's protocol. Thesequencing reactions were purified by binding to magnetic streptavidincoated beads (Detach Streptavidin Beads, Nordiag, cat. no. 2012-01)using a Magnatrix 8000 (Magnetic Biosolution), and analyzed on ABI(PRISM) 3130xl Genetic Analyzer (PE Applied Biosystems).

Production of Z Variants for ELISA:

Sequenced Z variants were produced by inoculating single colonies fromthe selections into 10 ml TSB-YE medium supplemented with 100 μg/mlampicillin and 0.1 mM IPTG and incubating for 24 h at 37° C. Cells werepelleted by centrifugation, re-suspended in 2 ml PBST (PBS supplementedwith 0.05% Tween-20), frozen at −80° C. and thawed in a water bath, torelease the periplasmic fraction of the cells. The freeze-thawingprocedure was repeated seven times and cells were then pelleted bycentrifugation. The supernatant of the periplasmic extract contained theZ variants as fusions to ABD, expressed as AQHDEALE-[Z#####]-VDYV-[ABD]-YVPG (SEQ ID NO: 1100) (Grönwall et al., supra). Z##### refers to individual, 58 amino acid residue Z variants.

ELISA K_(D) Analysis of Z Variants:

The binding of Z variants to FcRn was analyzed in ELISA assays.Half-area 96-well ELISA plates were coated with 2 μg/ml of an anti-ABDgoat antibody (produced in-house) diluted in coating buffer (50 mMsodium carbonate, pH 9.6) at 4° C. overnight. The antibody solution waspoured off and the wells were blocked with 100 μl of PBSC (PBSsupplemented with 0.5% casein) for 1.5 h at RT. The blocking solutionwas discarded and 50 μl periplasmic solution, diluted 1:4, was added tothe wells and incubated for 1.5 h at RT under slow shaking. Thesolutions were poured off and the wells were washed four times witheither 0.05% PCT buffer, pH 6.0 (Mcllvaines phosphate-citrate buffer, pH6.0, supplemented with 0.05% Tween-20) or 0.05% PCT buffer, pH 7.4(Mcllvaines phosphate-citrate buffer, pH 7.4, supplemented with 0.05%Tween-20). The target protein, biotinylated human FcRn, was added to thewells in a 1:3 diluted concentration series from 2 μg/ml (45 nM) to 0.3ng/ml (6.9 μM) diluted in PCC buffer, pH 6.0 or pH 7.4, (Mcllvainesphosphate-citrate buffer, pH 6.0 or pH 7.4, supplemented with 0.5%casein), respectively. The plates were incubated for 1.5 h at RTfollowed by washes as described above. Streptavidin conjugated HRP(Thermo Scientific, cat. no. N100) was diluted 1:30 000 in PCC buffer,pH 6.0 or pH 7.4, respectively, and added to the wells followed by 45min incubation. After washing as described above, 50 μl ImmunoPure TMBsubstrate (Thermo Scientific, cat. no. 34021) was added to the wells andthe plates were treated according to the manufacturer's recommendations.Absorbance was measured at 450 nm using a multi-well plate reader,Victor³ (Perkin Elmer). A Z variant binding an irrelevant protein wasused as negative control and a blank was created by omitting theperiplasmic step. A Z variant which bound to FcRn in a pre-experiment(Z07918, SEQ ID NO:707) was used as positive control. Measured valueswere analyzed using GraphPad Prism 5 (GraphPad Software, LaJolla,Calif., USA) and non-linear regression in order to determine theaffinities (K_(D)) of the interactions.

ELISA Specificity Analysis of Z Variants:

In another ELISA experiment, the specificities of the Z variants weretested by assaying them against 2 μg/ml biotinylated human proteins B2M,PSMA (in house produced) and IgG (polyclonal, Pharmacia, Sweden) andagainst PCC buffer pH 6.0 or pH 7.4, respectively. The assay wasperformed at pH 6.0 and at pH 7.4, respectively, as described above. Thebiotinylated proteins or buffer were added to the wells instead of FcRnin the target protein step.

Results

Phage Display Selection of FcRn Binding Z Variants:

Individual clones were obtained after four cycles of phage displayselections against biotinylated human FcRn.

Sequencing:

Sequencing was performed on clones picked at random from selection roundfour. Each Z variant was given a unique identification number ##### andindividual variants are referred to as Z #####. The amino acid sequencesof the 58 amino acid residues long Z variants are listed in FIG. 1A-1MMas SEQ ID NO:707-722 and SEQ ID NO:1059. The deduced FcRn binding motifsof these Z variants are listed in FIG. 1A-1MM as SEQ ID NO:1-16 and SEQID NO:353. The amino acid sequences of the 49 amino acid residues longpolypeptides predicted to constitute the complete three-helix bundlewithin each of these Z variants are listed in FIG. 1A-1MM as SEQ IDNO:354-369 and SEQ ID NO:706.

ELISA Assays with Z Variants:

Sixteen clones were produced as ABD fusion proteins in E. coli. Theperiplasmic fractions were used in an ELISA against a dilution series ofhuman FcRn. The clones were: Z07909 (SEQ ID NO:719), Z07918 (SEQ IDNO:707), Z07930 (SEQ ID NO:712), Z07960 (SEQ ID NO:710), Z10109 (SEQ IDNO:709), Z10111 (SEQ ID NO:714), Z10127 (SEQ ID NO:718), Z10129 (SEQ IDNO:715), Z10140 (SEQ ID NO:711), Z10141 (SEQ ID NO:716), Z10145 (SEQ IDNO:721), Z10152 (SEQ ID NO:720), Z10156 (SEQ ID NO:717), Z10161 (SEQ IDNO:722), Z10183 (SEQ ID NO:713) and Z10193 (SEQ ID NO:708). K_(D) valueswere determined for all variants at pH 6.0 and for three variants at pH7.4 (Table 1). For thirteen variants, data was not obtained for a K_(D)analysis at pH 7.4. None of the sixteen variants displayed non-specificbinding when assayed against human B2M, IgG or PSMA.

TABLE 1 ELISA K_(D) analysis of Z-ABD variants in E. coli periplasmicfractions. Z variant SEQ ID NO: K_(D) pH 6.0 (M) K_(D) pH 7.4 (M) Z07909719 24.5 × 10⁻⁹  n.d. Z07918 707 2.0 × 10⁻⁹ 10.9 × 10⁻⁹ Z07930 712 10.4× 10⁻⁹  n.d. Z07960 710 6.0 × 10⁻⁹ n.d. Z10109 709 3.9 × 10⁻⁹ 23.9 ×10⁻⁹ Z10111 714 11.4 × 10⁻⁹  n.d. Z10127 718 21.3 × 10⁻⁹  n.d. Z10129715 17.6 × 10⁻⁹  n.d. Z10140 711 8.8 × 10⁻⁹ n.d. Z10141 716 21.2 × 10⁻⁹ n.d. Z10145 721 42.0 × 10⁻⁹  n.d. Z10152 720 24.6 × 10⁻⁹  n.d. Z10156717 21.3 × 10⁻⁹  n.d. Z10161 722 163.0 × 10⁻⁹  n.d. Z10183 713 10.9 ×10⁻⁹  n.d. Z10193 708 2.3 × 10⁻⁹ 25.9 × 10⁻⁹ n.d. = not determinable

Example 3 Production and Characterization of FcRn Binding Z Variants

In this Example, seventeen Z variants were produced in E. coli, purifiedand assayed against human FcRn in Biacore. A subset of said variants wasalso assayed against mouse FcRn. Circular dichroism (CD) spectroscopywas performed for a subset of Z variants for investigation of theirsecondary structure.

Materials and Methods

Subcloning of Z Variants:

The DNA of seventeen FcRn binding Z variants (SEQ ID NO:707-722 and SEQID NO:1059) was amplified from the library vector pAY02592. A subcloningstrategy for construction of monomeric Z variant molecules withN-terminal His₆ tag was applied using standard molecular biologytechniques (essentially as described in detail in WO2009/077175 for Zvariants binding another target). The Z gene fragments were subclonedinto the expression vector pAY01448 resulting in the encoded sequenceMGSSHHHHHHLQ-[Z #####]-VD (SEQ ID NO: 1101).

In addition, the FcRn binding variant Z07918 (SEQ ID NO:707), butstarting with the amino acids AE instead of VD and denoted Z11948 (SEQID NO:1060), was cloned as homodimeric constructs with two differentlinkers between the Z variants and followed by a C-terminal His₆ tag.This was performed using conventional molecular biology methodsincluding DNA amplification, restriction with suitable restrictionenzymes and ligation of the DNA. The two linkers were obtained fromThermo Fisher Scientific. The Z gene fragments were subcloned into theexpression vector (pET-26 origin, Novagen) resulting in the encodedsequence [Z #####]-GT-(G₄S)-PR-[Z #####]-LEHHHHHH (SEQ ID NO: 1102) and[Z #####]-GT-(G₄S)₃-[Z #####]-LEHHHHHH (SEQ ID NO: 1103), respectively.

Cultivation and purification: E. coli BL21(DE3) cells (Novagen) weretransformed with plasmids containing the gene fragment of eachrespective FcRn binding Z variant and cultivated at 37° C. in 800 or1000 ml of TSB-YE medium supplemented with 50 μg/ml kanamycin. AtOD₆₀₀=2, IPTG was added to induce expression at a final concentration of0.17 or 0.2 mM and the culture was incubated at 37° C. for another 5 h.The cells were harvested by centrifugation.

Approximately 2-5 g of each cell pellet was resuspended in 10-25 mlbinding buffer (20 mM sodium phosphate, 0.5 M NaCl, 20 mM imidazole, pH7.4) supplemented with BENZONASE (Merck, cat. no. 1.01654.0001) to aconcentration of 15 U/ml and Lysozyme (Sigma, cat. no. L-7651) to aconcentration of 0.5 mg/ml. After cell disruption by threefreeze-thawing cycles or sonication, cell debris was removed bycentrifugation and each supernatant was applied on a 1 ml His GraviTrapIMAC column (GE Healthcare, cat. no. 11-0033-99). Contaminants wereremoved by washing with wash buffer (20 mM sodium phosphate, 0.5 M NaCl,20 or 60 mM imidazole, pH 7.4), and the FcRn binding Z variants weresubsequently eluted with elution buffer 1 (20 mM sodium phosphate, 0.5 Msodium chloride, 250 mM imidazole, pH 7.4) or elution buffer 2 (0.1 Macetic acid, 0.5 M sodium chloride, pH 4.5). Purified Z variants werebuffer exchanged to PBS using PD-10 columns (GE Healthcare), accordingto the manufacturer's protocol. Protein concentrations were determinedby measuring the absorbance at 280 nm, using a NANODROP ND-1000spectrophotometer, and using the extinction coefficient of therespective protein. The purity of the FcRn binding Z variants wasanalyzed by SDS-PAGE stained with Coomassie Blue. The identity of eachpurified FcRn binding Z variant was confirmed using LC/MS analysis.

Cd Analysis:

Purified His₆-tagged Z variants were diluted to 0.5 mg/ml in PBS. Foreach diluted Z variant, a CD spectrum at 250-195 nm or 250-190 nm wasobtained at 20° C. In addition, a variable temperature measurement (VTM)was performed to determine the melting temperature (Tm). In the VTM, theabsorbance was measured at 221 nm while the temperature was raised from20 to 90° C., with a temperature slope of 5° C./min. A new CD spectrumwas obtained at 20° C. after the heating procedure in order to study therefolding ability of the Z variants. The CD measurements were performedon a Jasco J-810 spectropolarimeter (Jasco Scandinavia AB) using a cellwith an optical path-length of 1 mm.

Biacore Binding and Kinetic Analysis:

The interaction of FcRn binding His₆-tagged Z variants with human FcRnwas analyzed in a Biacore 2000 instrument (GE Healthcare). Human FcRnwas immobilized in a flow cell on the carboxylated dextran layer of aCM5 chip surface (GE Healthcare). The immobilization was performed usingamine coupling chemistry according to the manufacturer's protocol andusing HBS-EP (GE Healthcare) as running buffer. One flow cell surface onthe chip was activated and deactivated for use as blank during analyteinjections. In the two binding experiments presented below, Mcllvainesphosphate-citrate buffer pH 6.0 supplemented with 0.005% Tween-20(0.005% PCT) was used as running buffer. In all experiments, a flow rateof 50 μl/min was used.

In one experiment, the dissociation at pH 6.0 was compared to thedissociation at pH 7.4. His₆-tagged Z variants and a human monoclonalIgG1 were diluted in running buffer to a final concentration of 250 nMor 2.5 nM, respectively, and injected over the FcRn chip for 1 minuteusing the co-inject procedure. The second injection of the co-injectprocedure, representing the dissociation phase of the interactions,contained either running buffer (pH 6.0) or 0.005% PCT pH 7.4. The Zvariants were allowed to dissociate for 1 minute, except for Z07918 andZ10193, which were allowed to dissociate for 4 minutes, before a surfaceequilibration during 5 minutes in running buffer. IgG was allowed todissociate for 4 minutes before equilibration. Buffer injections wereperformed in a similar way; co-injection of buffer pH 6.0 followed by pH6.0 or co-injection of buffer pH 6.0 followed by pH 7.4. The resultswere analyzed in BiaEvaluation software 4.1 (GE Healthcare). Curves ofthe blank surface were subtracted from the curves of the ligand surface.In addition, curves of buffer injections were subtracted from the Zvariant curves and from the IgG curves to adjust for the buffer effects.

In another experiment, approximate kinetic constants (k_(on) andk_(off)) and affinities (K_(D)) were determined for a subset ofHis₆-tagged Z variants. Three concentrations of the Z variants wereinjected for 1 minute followed by dissociation in running buffer for 1minute. The surfaces were equilibrated with running buffer during 7.5minutes before the start of next cycle. Injected concentrations wereeither 675 nM, 225 nM and 75 nM (Z10140, Z10156 and Z10183) or 225 nM,75 nM and 25 nM (Z07918 and Z10193). Kinetic constants were calculatedfrom the sensorgrams using the Langmuir 1:1 model of BiaEvaluationsoftware 4.1 (GE Healthcare).

In a separate experiment, the affinity of the interactions of Z variantsto hFcRn (SEQ ID NO:1065) and mFcRn (SEQ ID NO:1070), respectively, wasmeasured at both pH 6.0 and pH 7.4 on a Biacore 3000 instrument (GEHealthcare). hFcRn and mFcRn were produced essentially as described inExample 1 but using mouse 3T3 cells instead of human SKOV-3 cells forproduction of mFcRn, and immobilized on separate flow cells on a CM5chip in acetate buffer at pH 4.65. The immobilization level wasapproximately 1000 RU for both receptors. A reference flow cell wascreated by activation and deactivation. 0.005% PCT pH 6.0 or 7.4 wasused as running buffer and for dilution of the analytes. All analyseswere performed at 25° C. The affinity constants for the His₆-tagged Zvariants Z07918 (SEQ ID NO:707), Z07960 (SEQ ID NO:710) and Z10193 (SEQID NO:708) were determined by injecting a dilution series from 1024 nMto 0.5 nM (pH 6.0) or from 10240 nM to 5 nM (pH 7.4). The affinitieswere derived using GraphPad Prism 5 software, using a one site bindingsaturation model.

AlphaLISA Blocking Assay:

The potential of Z variants to inhibit binding of IgG to FcRn wasanalyzed in an AlphaLISA assay with an EnSpire multiplate reader 2300(Perkin Elmer). Human IgG (Roactemra) was immobilized on AlphaLISAacceptor beads (Perkin Elmer, cat. no. 6772002) according to themanufacturer's recommendations. Stepwise serial dilutions 1:3 ofHis-tagged Z variants to final concentrations of 250 nM to 38 μM weremade in a 384-well plate (Perkin Elmer, cat. no. G6005350) and incubatedfor 45 min with 10 nM biotinylated human FcRn (Biorbyt, cat. no.orb84388, biotinylated essentially as described in Example 2) inAlphaLISA buffer (Perkin Elmer, cat. no. AL000F) adjusted to pH 6.0using HCl. IgG-coated Acceptor beads were added to a final concentrationof 10 μM and incubated for 45 min. Finally, streptavidin coated Donorbeads (Perkin Elmer, cat. no. 6772002) were added to a finalconcentration of 40 μg/ml and incubated for 30 min. All incubations wereperformed at RT in the dark. The plate was analyzed in the EnSpireinstrument and the IC50 values were calculated using GraphPad Prism 5.

Results

Cultivation and Purification:

The seventeen FcRn binding Z variants (SEQ ID NO: 707-722 and SEQ IDNO:1059), constructed with an N-terminal His₆ tag, were produced in E.coli. The amount of IMAC-purified protein from approximately 2-5 gbacterial pellets, determined spectrophotometrically by measuring theabsorbance at 280 nm, ranged from approximately 10 mg to 20 mg for thedifferent FcRn binding Z variants. SDS-PAGE analysis of each finalprotein preparation showed that these predominantly contained the FcRnbinding Z variant. The correct identity and molecular weight of eachFcRn binding Z variant was confirmed by HPLC-MS analysis.

Cd Analysis:

The CD spectra determined for six Z variants showed that each had anα-helical structure at 20° C. This result was also verified in thevariable temperature measurements, wherein melting temperatures (Tm)were determined (Table 2). A reversible folding was seen for the six Zvariants when overlaying spectra measured before and after heating to90° C.

TABLE 2 Melting temperatures for a selection of Z variants. Z variantSEQ ID NO: Tm (° C.) Z07909 719 56 Z07918 707 49 Z07930 712 56 Z07960710 58 Z10109 709 61 Z10193 708 59

Biacore Binding and Kinetic Analyses:

The binding of seventeen Z variants to human FcRn and the dissociationat different pH were tested in a Biacore instrument by sequentiallyinjecting each of the Z variants at pH 6.0 and either buffer pH 6.0 orpH 7.4 over a chip surface containing FcRn. The ligand immobilizationlevel of the surface was 1668 RU human FcRn. The seventeen Z variantsshowed binding to FcRn at pH 6.0, and for all variants, faster off-rateswere seen at pH 7.4 compared to pH 6.0. The result for IgG was similar,displaying a faster off-rate at pH 7.4. The variants Z07918 and Z10193showed the slowest dissociation curves. Sensorgrams for a subset ofvariants and IgG are displayed in FIG. 2 A-E.

TABLE 3 Biacore kinetic constants and affinities for hFcRn binding at pH6.0. Z variant SEQ ID NO: k_(on) (M⁻¹s⁻¹) k_(off) (s⁻¹) K_(D) (M) Z07918707 1.4 × 10⁶  0.022 1.6 × 10⁻⁸ Z10140 711 1.4 × 10⁶ 0.12 8.6 × 10⁻⁸Z10156 717 7.6 × 10⁵ 0.28 3.7 × 10⁻⁷ Z10183 713 1.0 × 10⁶ 0.13 1.3 ×10⁻⁷ Z10193 708 1.5 × 10⁶  0.033 2.2 × 10⁻⁸

The kinetic constants of five Z variants interacting with FcRn at pH 6.0were determined (see Table 3). The immobilization level of the surfacewas 2015 RU human FcRn. For each Z variant, kinetic constants werecalculated using a curve set of three injected concentrations.

Affinity (K_(D)) constants were also determined for His₆-tagged Zvariants Z07918 (SEQ ID NO:707), Z07960 (SEQ ID NO:710) and Z10193 (SEQID NO:708) interacting with human and mouse FcRn at pH 6.0 and pH 7.4(Table 4). For all three variants, K_(D) values were lower at pH 6.0compared to pH 7.4.

TABLE 4 Biacore affinities for hFcRn and mFcRn at pH 6.0 and pH 7.4. SEQID K_(D) (M) hFcRn K_(D) (M) mFcRn Z variant NO: pH 6.0 pH 7.4 pH 6.0 pH7.4 Z07918 707 1.2 × 10⁻⁸ >5 × 10⁻⁷ 9.0 × 10⁻⁸ >5 × 10⁻⁷ Z07960 710 5.0× 10⁻⁸ >1 × 10⁻⁶ 3.5 × 10⁻⁷ >5 × 10⁻⁶ Z10193 708 1.4 × 10⁻⁸ >5 × 10⁻⁷9.5 × 10⁻⁸ >5 × 10⁻⁷

TABLE 5 Calculated IC50 values from AlphaLISA blocking assay. SEQ ID NOof IC50 Z variant Z variant (M) Z07909 719 4.6 × 10⁻⁸ Z07918 707 2.1 ×10⁻⁹ Z07930 712 4.2 × 10⁻⁸ Z07960 710 4.2 × 10⁻⁸ Z10109 709 5.7 × 10⁻⁸Z10111 714 4.6 × 10⁻⁸ Z10140 711 5.6 × 10⁻⁸ Z10183 713 3.9 × 10⁻⁸ Z10193708 1.2 × 10⁻⁸ Z13993 1059  1.3 × 10⁻⁷ Z11948-G₄S-Z11948 1060   3.8 ×10⁻¹⁰ Z11948-(G₄S)₃-Z11948 1060   4.1 × 10⁻¹⁰

AlphaLISA Blocking Assay:

The ability of seventeen His₆-tagged monomeric Z variants (SEQ IDNO:707-722 and SEQ ID NO:1059) and two dimeric variant,Z11948-G₄S-Z11948 and Z11948-(G₄S)₃-Z11948 to inhibit IgG binding toFcRn was tested in an AlphaLISA blocking assay. Serial dilutions of theZ variants were incubated with biotinylated human FcRn and the blockingability of each respective variant was measured after addition of IgGcoated Acceptor beads and subsequently streptavidin coated Donor beads.Inhibition could be measured as a decrease in AlphaLISA counts forpositive Z variants. The calculated IC50 values for the ten monomericvariants and the two dimeric variants that were shown to block IgGbinding to FcRn in this assay are shown in Table 5.

Example 4 Binding of FcRn Binding Z Variants to Human or Mouse FcRn/eGFPTransfected HeLa Cells

In this example, the binding ability of FcRn binding Z variants wasinvestigated. The production of HeLa cells expressing human and murineFcRn-eGFP gene transgene and the use of these cells for flow cytometryanalysis with Alexa647 labeled Z variants is described.

Materials and Methods

Cloning of FcRn-eGFP and B2M Viral Vectors:

The genes encoding murine FcRn (mFcRn, Genbank BC003786.1,OpenBiosystems) and murine B2M (mB2M, Genbank BC085164.1,OpenBiosystems) were amplified in a similar way as the genes for humanFcRn and human B2M as described in Example 1. Human and murine FcRn andB2M genes were amplified as follows: for hFcRn, the sequence encodingamino acids 1-365 (SEQ ID NO:1068) was amplified; for hB2M, the sequenceencoding amino acids 21-119 (SEQ ID NO:1066) was amplified; for mFcRn,the sequence encoding amino acids 1-369 (SEQ ID NO:1069) was amplified;and for mB2M, the sequence encoding amino acids 21-119 (SEQ ID NO:1067)was amplified. The vector pHR-cPPT-CMV-EGFP (Jakobsson et al. (2003) JNeurosci Res 73:876-85) and FcRn PCR amplicons (human and murine) werecut using the restriction enzymes BamHI (human) or BclI (murine) andMluI (New England Biolabs, cat. nos. R0136M, R0160L and R0198L,respectively), and ligated using T4 DNA Ligase (New England Biolabs,cat. no. M0202M). The ligation mix was chemically transformed into E.coli RRIΔM15 and spread on ampicillin plates. Colonies were picked andscreened with suitable primer pairs. The construct encoding the originalsignal peptide, human or murine FcRn and eGFP at the cytoplasmic tailwere verified by sequencing and denoted pHR-cPPT-CMV-hFcRn-eGFP andpHR-cPPT-CMV-mFcRn-eGFP, respectively.

The human and murine B2M PCR amplicons were inserted into the plasmidpDONOR221 (Invitrogen, cat. no. 12536-017) by recombination using theGateway system (Invitrogen, cat. no. 11789020, GATEWAY BP CLONASE IIEnzyme mix) according to the manufacturer's recommendations. Afterverification of correct sequences, human or murine B2M was inserted intop2k7_gtc (Suter et al., supra) using a multi-site gateway cloning system(Invitrogen, cat. no. 11791020, GATEWAY LR CLONASE II Enzyme mix)together with the promoter containing plasmid pENTR-CMV (Tai et al.supra), resulting in the vectors 2k7_(neo)-CMV-hB2M and2k7_(neo)-CMV-mB2M, respectively.

Lentiviral Transduction of HeLa Cells:

The vector pairs 2k7_(neo)-CMV-hB2M and pHR-cPPT-CMV-hFcRn-eGFP or2k7_(neo)-CMV-mB2M and pHR-cPPT-CMV-mFcRn-eGFP were co-transfectedtogether with VSV-G envelope and gag/pol packaging plasmid into HEK293Tcells using calcium chloride transfection (Zufferey et al., supra;Jakobsson et al. (2006) supra). HEK293T culture supernatants containingformed lentiviral particles with FcRn and B2M transgenes respectivelywere used to sequentially transduce HeLa Cervix adenocarcinoma cells(Cell Line Service) at low passage number. The resulting two stablytransduced HeLa cell lines are in the following denoted hFcRn-eGFP(transduced with genes for human FcRn-eGFP and hB2M) and mFcRn-eGFP(transduced with genes for mouse FcRn-eGFP and mB2M).

Alexa647 Labeling of FcRn Binding Z Variants:

The three His₆-tagged Z variants Z07918, Z07930 and Z07960 were labeledwith ALEXA FLUOR 647 Carboxylic Acid Succinimidyl Ester (Invitrogen cat.no. A20106). Before labeling, buffer was exchanged to 0.2 M carbonatebuffer, pH 8.3, using Vivaspin500 centrifugal filter units (10 kDa MWCO,Vivaproducts cat. no. 512-2838) spun at 10,000 g. The labeling wasperformed in the Vivaspin500 and 1 μl of Alexa647 Succinimidyl Ester dye(40 μg/μl in DMSO corresponding to 1.3× molar excess) was added to 200μg/25 μl Z variant. The mixes were incubated at RT in the dark for 40minutes in a wiggling rota mixer. The reaction mixes were subsequentlyput on ice for 3.5 hours and free dye was removed by washing with 15×100μl PBS in the Vivaspin500.

Immunofluorescence Staining of Human and Mouse FcRn-eGFP TransfectedHeLa-Cells with FcRn Binding Z Variants:

hFcRn-eGFP and mFcRn-eGFP HeLa cells were harvested by trypsination andwashed twice in PBS at pH 6.0 before counting. 100,000 cells werepipetted per well of a v-bottomed 96 well plate (Nunc, cat no 277143)and the cells in the plate were subsequently pelleted at 1,700 rpm for 4min at 4° C. The supernatants were removed and the cells were fixed with50 μl of 2% formaldehyde (Sigma Aldrich, cat. no. F8775) in PBS at pH6.0 for 10 min at RT. Cells were thereafter washed with 2×100 μl PBS pH6.0, saturated with casein (PBSC), and resuspended in PBSC plus 0.1%saponin (AppliChem, cat no A4518.0100) containing 620 nM of Alexa647labeled His₆-tagged Z variants; Z07960, Z07930 and Z07918. TransducedHeLa cells, incubated with buffer alone, were used as control. The cellswere incubated for 1 h at 8° C. on a shaker in the dark, washed with2×100 μl PBSC and resuspended in 180 μl of PBS pH 6.0 plus 1% BSA(fraction V, Merck, cat. no. 1.12018.0100). 10,000 cells/well wereanalyzed in a Gallios Flow Cytometer (Beckman Coulter) and the data wasanalyzed using Kaluza software (Beckman Coulter).

Results

Flow cytometry analysis was utilized to determine whether the FcRnbinding Z variants could bind to human and/or mouse FcRn on human ormouse FcRn/eGFP transduced HeLa cells. The experiment was performed atpH 6.0 with Alexa647 labeled Z07960, Z07930 and Z07918 (SEQ ID NO:710,712 and 707, respectively). Dot plot analysis (y-axis: Alexa647, x-axis:eGFP) showed that the transduced cell population could be divided intoFcRn-eGFP negative and positive population (FIG. 3, gate H and I,respectively) indicating heterogeneous expression of the FcRn-eGFPfusion protein by HeLa cells (FIG. 3). Accordingly, the meanfluorescence intensity (MFI) values for Alexa647 in gate I weresubtracted by background MFI values of Alexa647 in gate H. Thecalculated MFI values are presented in FIG. 4. The results show thatZ07960, Z07930 and Z07918 are capable of binding HeLa cells displayinghuman (FIG. 4, Panel A) or murine (FIG. 4, Panel B) FcRn-eGFP.

Example 5 Blocking of IgG Binding to FcRn with the FcRn Binding ZVariant Z07918

In this example, the potential competition of FcRn binding Z variantswith IgG for binding to FcRn was investigated in a cell based assay.Such binding will result in blocking of the IgG-FcRn interaction.

Materials and Methods

Blocking of IgG-FcRn Immunofluorescence Staining:

Human or murine FcRn-eGFP transduced HeLa cells were prepared asdescribed in Example 4. Fixed cells were resuspended in 50 μl of a mixof either 100 nM Alexa647-conjugated human or mouse IgG (Jacksonlaboratories, cat. no. 009-600-003 and 015-600-003, respectively) and1000, 100, 10, 1 or 0 (buffer control) nM His₆-tagged Z07918 diluted inPBS-casein, pH 6.0, plus 0.1% saponin (AppliChem). The cells wereincubated for 1 h at 37° C. on a shaker in the dark, washed with 2×100μl PBS-casein pH 6.0 and re-suspended in 180 μl of PBS, pH 6.0, plus 1%BSA. Data from 10,000 cells/well (except somewhat fewer cells for mouse100 nM mIgG-Alexa647) were obtained using a Gallios Flow Cytometer(Beckman Coulter) and the data was analyzed using Kaluza software(Beckman Coulter).

Results

The experiment was performed to determine if the FcRn binding Z variantZ07918 (SEQ ID NO:707) blocks the IgG-FcRn interaction. Human or murineFcRn-eGFP transduced HeLa cells were incubated with human or mouseAlexa647-conjugated IgG. The binding was blocked with unlabeled Z07918at different concentrations. Due to the heterogeneous expression of FcRnby the transduced HeLa cells (described in Example 4), the MFI valuesfor Alexa647 in gate N of each sample was subtracted by thecorresponding MFI values in gate M (FIG. 5). The percent IgG Alexa647binding was calculated by dividing the different MFI values with the MFIfor the blank control. The results showed that Z07918 effectivelyblocked hIgG binding to hFcRn (FIG. 6, Panel A) in a dose dependentmanner. Furthermore, Z07918 also blocked mIgG binding to mFcRn (FIG. 6,Panel B) although less efficiently compared to hIgG-binding.

Example 6 Pharmacokinetic Study of Three FcRn Binding Z Variants

In this example, the ability of FcRn binding Z variants to prolong serumhalf-life of a non-specific Z variant was investigated by apharmacokinetic study performed in mice.

Materials and Methods

Subcloning of Z Variants:

A subset of Z variants (Z07918, Z07960 and Z10193) was submitted to asecond subcloning. DNA from the subcloned His₆-tagged variants inExample 3 was used as template. First, PCR amplification using suitableprimer pairs was performed to create genes encoding Z variants startingwith the amino acids AE instead of VD. The mutated Z variants are listedin FIG. 1A-1MM and were denoted Z11948 (SEQ ID NO:1060), Z11946 (SEQ IDNO:1061) and Z11947 (SEQ ID NO:1062), corresponding to mutated Z07918,Z07960 and Z10193, respectively. Genes encoding the new Z variants wererestriction cleaved and ligated into a vector harboring the genesencoding albumin binding variant PP013 (SEQ ID NO:1063) and Z03638 (SEQID NO:1064) with spacer sequences resulting in a gene fusion encoding [Z#####]-GAP(G4S)₄TS-[PP013]-GT(G₄S)₄PR-[Z03638] (SEQ ID NO: 1104) (alsodenoted “Z #####-PP013-Z03638” or “Z variant in fusion withPP013-Z03638”). The negative control molecule[Z03638]-GAP(G₄S)₄TS-[PP013] (SEQ ID NO: 1105) was subcloned in asimilar way by ligating Z03638 into a vector containing a (G₄S)₄ linkerand the sequence for PP013. The subsequent steps for vectortransformation into E. coli were performed as in Example 3.

Cultivation and Purification:

Z variants in fusion with PP013-Z03638 were produced in E. coli asdescribed in Example 3. Approximately 3 g of each cell pellet wasre-suspended in 30 ml TST-buffer (25 mM Tris-HCl, 1 mM EDTA, 200 mMNaCl, 0.05% Tween20, pH 8.0) supplemented with BENZONASE (Merck). Aftercell disruption by sonication and clarification by centrifugation, eachsupernatant was applied on a gravity flow column with 5 ml agaroseimmobilized with an anti-ABD ligand (produced in-house). After washingwith TST-buffer and 5 mM NH₄Ac buffer, pH 5.5, the Z variants wereeluted with 0.1 M HAc. Acetonitrile (ACN) was added to a finalconcentration of 10% to the eluted fractions from the anti-ABD agaroseaffinity chromatography purification step and the samples were loaded ona 3 ml Resource 15RPC column (GE Healthcare), previously equilibratedwith RPC solvent A (0.1% trifluoroacetic acid (TFA), 10% ACN, 90%water). After column wash with RPC solvent A, bound protein was elutedwith a linear gradient 0-50% RPC solvent B (0.1% TFA, 80% ACN, 20%water) during 60 ml. Fractions containing pure Z variant were identifiedby SDS-PAGE analysis and pooled. After the RPC purification, the bufferof the pools was exchanged to PBS using a HiPrep 26/10 Desalting column(GE Healthcare). Finally, the Z variants were purified on 1 ml EndoTrapred columns (Hyglos, cat. no. 321063) to ensure low endotoxin content.

Protein concentrations, purities and the identity of each purified Zvariant were analyzed as described in Example 3.

Biacore Analysis:

Expressed and purified Z variants fused to PP013-Z03638 were assayedagainst human FcRn at pH 6.0 essentially as described for the kineticanalysis in Example 3. The Z variants and the negative controlZ03638-PP013 were injected at 40 nM, 160 nM and 640 nM during 1 minutefollowed by dissociation for 2.5 minutes and equilibration for 1 minute.Kinetic constants and affinities were determined for the Z variantsusing the BiaEvaluation software.

Pharmacokinetic Study:

Z11947, Z11946 and Z11948 fused to PP013-Z03638 were administeredintravenously (i.v.) to male NMRI mice (Charles River, Germany) at adose of 92 nmol/kg body weight. Sera from groups of three mice wereobtained at 0.08, 6, 18, 78, 120, 168 and 240 hours. The concentrationof respective Z variant was determined by ELISA.

ELISA:

Half-area 96-well ELISA plates were coated at 4° C. overnight with 50μl/well of an Z specific goat antibody (produced in-house) diluted to 4μg/ml in coating buffer (50 mM sodium carbonate, pH 9.6). The antibodysolution was poured off and the wells were blocked with 100 μl of PBSCfor 1.5 h at RT. The sera were diluted in PBSC plus 1% mouse serum(matrix) from 1:100 to 1:51,200 in a two-fold dilution series in adilutions plate. A standard titration for respective Z variant and fourquality controls (very low, low, medium and high control) diluted inmatrix were included on each plate. 50 μl of the dilutions weretransferred per well and the ELISA plates were incubated for 1.5 h atRT. The plates were washed four times with PBST. Bound Z variants weredetected with 50 μl/well of rabbit anti-PP013 Ig (produced in-house)diluted to 4 μg/ml in PBSC. The plates were subsequently incubated for1.5 h at RT followed by washes as described above. HRP conjugated donkeyanti-rabbit HRP obtained from Jackson laboratories (cat. no.711-035-152), diluted 1:20,000 in PBSC, was added and the plates wereincubated for 1 hour. After washing as described above, 50 μl ofImmunoPure TMB substrate was added to the wells and the plates weredeveloped according to the manufacturer's recommendations. After 15minutes of development, the absorbance was measured at 450 nm using amulti-well plate reader (Victor³). The absorbance values were analyzedusing GraphPad Prism 5 to determine the concentrations (cubic-splinecurve fit) and area under curve (AUC). The concentrations were thenplotted as their natural logarithms against time. The resulting curvesfollowed a two compartment model and the terminal half-life wascalculated as In2 divided by the slope based on the last three timepoints.

Results

Cultivation and Purification:

The three FcRn binding Z variants Z11947, Z11946 and Z11948 (SEQ IDNO:1062, 1061 and 1060), constructed as Z #####-PP013-Z03638, and thenegative control Z03638-PP013, were produced in E. coli. The amount ofpurified protein from approximately 3 g bacterial pellets, determinedspectrophotometrically by measuring the absorbance at 280 nm, rangedfrom approximately 10 to 25 mg for the different FcRn binding Zvariants. SDS-PAGE analysis of each final protein preparation showedthat they predominantly contained respective FcRn binding Z variant. Thecorrect molecular weight of each FcRn binding Z variant was confirmed byLC/MS analysis.

TABLE 6 Kinetic constants and affinities for FcRn at pH 6.0 of Zvariants produced as fusions to PP013-Z03638. Z variant SEQ ID NO:k_(on) (M⁻¹s⁻¹) k_(off) (s⁻¹) K_(D) (M) Z11948 1060 7.73 × 105 0.047 6.2× 10⁻⁸ Z11946 1061 3.35 × 105 0.275 8.2 × 10⁻⁷ Z11947 1062 6.54 × 1050.064 9.8 × 10⁻⁸

Biacore Analysis:

The binding to FcRn was analyzed for the three PP013-Z03638 fused Zvariants. The immobilization level of the surface was 548 RU of humanFcRn. The resulting rough kinetic constants and affinities for thetarget binding at pH 6.0 are displayed in Table 6. Fitted curves aredisplayed in FIG. 7A-C. The negative control Z03638-PP013 was negativeagainst FcRn.

Pharmacokinetic Study:

The pharmacokinetic profiles of the above-mentioned constructs of Zvariants fused to PP013-Z03638 were compared to the negative controlZ03638-PP013 in a mouse pharmacokinetic study. In previous work, e.g. asdescribed in PCT application WO2009/016043, it is shown that ABD fusionproteins have a long half-life in serum, caused by ABD binding to serumalbumin. In accordance with the previous results, terminal half-life ofABD-fused Z variant molecule (Z03638-PP013) was approximately 43 hours,which is comparable to half-life of mouse albumin (35 hours). Theterminal half-lives of the constructs containing FcRn binding Z variantmolecule in addition to ABD were two- to three-fold longer (FIG. 8). Thecalculated terminal half-lives were 99 hours (Z11947), 69 hours (Z11946)and 58 hours (Z11948), suggesting that FcRn binding of the Z variantscontributed to the prolonged half-life.

Example 7 Design and Construction of a Maturation Library of FcRnBinding Z Variants

In this Example, a maturated library was constructed. The library wasused for selections of FcRn binding Z variants. Selections frommaturated libraries are usually expected to result in binders withincreased affinity (Orlova et al., (2006) Cancer Res 66(8):4339-48). Inthis study, randomized single stranded linkers were generated usingsplit-pool synthesis enabling incorporation of defined codons in desiredpositions in the synthesis.

Materials and Methods

Library Design:

The library was based on the sixteen sequences of the human FcRn bindingZ variants in Table 1 and further described in Examples 2-6. In the newlibrary, 13 variable positions in the Z molecule scaffold were biasedtowards certain amino acid residues, according to a strategy mainlybased on the binding motifs of the Z variants defined in SEQ IDNO:707-722. A DNA linker was generated using split-pool synthesiscontaining the 147 bp partially randomized helix 1 and 2 of the aminoacid sequence: 5′-AA ATA AAT CTC GAG GTA GAT GCC AAA TAC GCC AAA GAA NNNNNN NNN GCG NNN NNN GAG ATC NNN NNN TTA CCT AAC TTA ACC NNN NNN CAA NNNNNN GCC TTC ATC NNN AAA TTA NNN GAT GAC CCA AGC CAG AGC TCA TTA TTT A-3′(SEQ ID NO:1074, randomized codons are illustrated as NNN) flanked byrestriction sites XhoI and SacI, was ordered from DNA 2.0 (Menlo Park,Calif., USA). The theoretical distributions of amino acid residues inthe new library, including eight variable amino acid positions (9, 10,11, 13, 14, 24, 32 and 35) and five constant amino acid positions (17,18, 25, 27 and 28) in the Z molecule scaffold are given in Table 8. Theresulting theoretical library size is 5.3×10⁸ variants.

TABLE 7 Design of library for maturation. Amino acid No of position inthe Randomization amino Z variant (amino acid abbreviations) acidsProportion 9 A,D,E,F,H,I,K,L,N,Q,R,S,T,V,W,Y 16 1/16 10A,D,E,F,H,I,K,L,M,N,Q,R,S,T,V,W,Y 17 1/17 11A,D,E,F,H,I,K,L,N,Q,R,S,T,V,W,Y 16 1/16 13A,D,E,F,G,H,I,K,L,N,Q,R,S,T,V,W,Y 17 1/17 14 A,F,H(25%),I,K,L,N,Q,R,S,T,V,W,Y 14 3/52, 13/52 (H) 17 R 1 1 18 W 1 1 24 F,Y2 1/2 25 D 1 1 27 R 1 1 28 V 1 1 32 A,D,E,F,H,I,K,L,N,Q,R,S,T,V,W,Y 161/16 35 A,D,E,F,H,I,K,L,N,Q,R,S,T,V,W,Y 16 1/16

Library construction: The library was amplified using AmpliTaq Goldpolymerase (Applied Biosystems, cat. no. 4311816) during 12 cycles ofPCR and pooled products were purified with QIAquick PCR Purification Kit(QIAGEN, cat. no. 28106) according to the supplier's recommendations.The purified pool of randomized library fragments was digested withrestriction enzymes XhoI and SacI-HF (New England Biolabs, cat. no.R0146L, and cat. no. R3156M) and concentrated using a PCR PurificationKit. Subsequently, the product was subjected to preparative 2.5% agarose(Nuisieve GTC agarose, Cambrex, Invitrogen) gel electrophoresis andpurified using QIAGEN gel extraction Kit (QIAGEN, cat. no. 28706)according to the supplier's recommendations.

The phagemid vector pAY02592 (essentially as pAffi1 described inGrönwall et al., supra) was restricted with the same enzymes, purifiedusing phenol/chloroform extraction and ethanol precipitation. Therestricted fragments and the restricted vector were ligated in a molarratio of 5:1 with T4 DNA ligase (Fermentas, cat. no. EL0011) for 2 hoursat RT, followed by overnight incubation at 4° C. The ligated DNA wasrecovered by phenol/chloroform extraction and ethanol precipitation,followed by dissolution in 10 mM Tris-HCl, pH 8.5. Thus, the resultinglibrary in vector pAY02592 encoded Z variants, each fused to an albuminbinding domain (ABD) derived from streptococcal protein G.

The ligation reactions (approximately 160 ng DNA/transformation) wereelectroporated into electrocompetent E. coli ER2738 cells (50 μl,Lucigen, Middleton, Wis., USA). Immediately after electroporation,approximately 1 ml of recovery medium (supplied with the ER2738 cells)was added. The transformed cells were incubated at 37° C. for 60 min.Samples were taken for titration and for determination of the number oftransformants. The cells were thereafter pooled and cultivated overnightat 37° C. in 1 l of TSB-YE medium, supplemented with 2% glucose, 10μg/ml tetracycline and 100 μg/ml ampicillin. The cells were pelleted for7 min at 4,000 g and resuspended in a PBS/glycerol solution(approximately 40% glycerol). The cells were aliquoted and stored at−80° C. Clones from the library of Z variants were sequenced in order toverify the content and to evaluate the outcome of the constructedlibrary vis-à-vis the library design. Sequencing was performed asdescribed in Example 1 and the amino acid distribution was verified.

Preparation of Phage Stock:

Phage stock containing the phagemid library was prepared in a 20 lfermenter (Belach Bioteknik). Cells from a glycerol stock containing thephagemid library were inoculated in 10 l of TSB-YE (Tryptic SoyBroth-Yeast Extract; 30 g/l TSB, 5 g/l yeast extract) supplemented with1 g/l glucose, 100 mg/l ampicillin and 10 mg/l tetracycline. When thecells reached an optical density at 600 nm (OD600) of 0.6, approximately1.5 l of the cultivation was infected using a 5× molar excess of M13K07helper phage. The cells were incubated for 30 min, whereupon thefermenter was filled up to 10 l with complex fermentation medium [2.5g/l (NH₄)₂SO₄, 5.0 g/l yeast extract; 30 g/l tryptone, 2 g/l K2HPO₄; 3g/l KH₂PO₄, 1.25 g/l, Na₃C₆H₅O₇.2 H₂O; Breox FMT30 antifoaming agent 0.1ml/l]. The following components were added: 10 ml carbenicillin 25mg/ml, 5 ml kanamycin 50 mg/ml, 1 ml 1 Misopropyl-β-D-1-thiogalactopyranoside (IPTG); 17.5 ml/l of 300 g/lMgSO₄, and 5 ml of a trace element solution [35 g/l FeCl₃. 6 H₂O; 10.56g/l ZnSO₄.7 H₂O; 2.64 g/l CuSO₄.5 H₂O; 13.2 g/l MnSO₄.H₂O; 13.84 g/lCaCl₂.2 H₂O, dissolved in 1.2 M HCl]. A glucose limited fed-batchcultivation was started where a 600 g/l glucose solution was fed to thereactor (3.5 g/h in the start, 37.5 g/h after 20 h and until the end ofthe cultivation). pH was controlled at pH 7 through the automaticaddition of 25% NH₄OH, air was supplemented (5 l/min), and the stirrerwas set at 500 rpm. After 24 h of fed-batch cultivation the OD600 was33.2. The cells in the cultivation were pelleted by centrifugation at15,900 g. The phage particles were precipitated from the supernatanttwice in PEG/NaCl, filtered and dissolved in PBS and glycerol as inExample 2. Phage stocks were stored at −80° C. until use in selection.

Results

Library Construction:

The new library was designed based on a set of 16 FcRn binding Zvariants with verified binding properties (Example 2-6). The theoreticalsize of the designed library was 5.3×10⁸ Z variants. The actual size ofthe library, determined by titration after transformation to E. coliER2738 cells, was 4.5×10⁹ transformants.

The library quality was tested by sequencing of 96 transformants and bycomparing their actual sequences with the theoretical design. Thecontents of the actual library compared to the designed library wereshown to be satisfying. A maturated library of potential binders to FcRnwas thus successfully constructed.

Example 8 Selection and Screening of Z Variants from a Maturated LibraryMaterials and Methods

Phage Display Selection of Matured FcRn Binding Z Variants:

The target proteins human FcRn (Biorbyt, cat. no. orb84388) and murineFcRn (Biorbyt, cat. no. orb99076) were biotinylated essentially asdescribed in Example 2 using biotin at 10× molar excess. Phage displayselections, using the new library of Z variant molecules described inExample 7, were performed in four cycles against human FcRn or murineFcRn essentially as in Example 2 but with the following exceptions.Selection buffers were 0.1% PCTG buffer, pH 5.5 (Mcllvainesphosphate-citrate buffer, pH 5.5, supplemented with 0.1% Tween-20 and0.1% gelatin) or 0.1% PCTG buffer, pH 7.4, (Mcllvaines phosphate-citratebuffer, pH 7.4, supplemented with 0.1% Tween-20 and 0.1% gelatin)respectively. Prior to selection, HSA was added to the selection buffersto a final concentration of 1.5 μM. All tubes and beads used in theselection were pre-blocked with either of the two different selectionsbuffers. A pre-selection step, by incubation of phage stock withSA-beads for 45 min, was performed in cycle 1. For capture ofphage-target complexes, 1 mg beads per 1.1 μg biotinylated human FcRn or1.6 μg biotinylated murine FcRn was used. Washes were performed with0.1% PCT buffer pH 5.5 or pH 7.4 except for tracks 2-1-2-1 and 2-1-2-2where 0.1% PCT supplemented with 25 nM IgG (HERCEPTIN) or 10 nM IgG,respectively, was used as outlined in Table 7.

The five tracks (1-5) in cycle 1 were divided in the second to fourthcycles, resulting in totally seven tracks (1-1 to 5-1) in cycle 2,eleven tracks (1-1-1 to 5-1-1) in cycle 3 and fourteen tracks (1-1-1-1to 5-1-1-1) in cycle 4. The bound phage particles were eluted asdescribed in Example 2.

An overview of the selection strategy, describing an increasedstringency in subsequent cycles, using a lowered target concentrationand an increased number of washes, is shown in Table 8.

TABLE 8 Overview of the maturation selection data. Phage stock TargetNumber Selection from library or Target conc. Selection Wash of Cycletrack selection track species (nM) pH pH washes 1 1 Zlib006FcRn.I human100 7.4 7.4 2 1 2 Zlib006FcRn.I human 100 7.4 5.5 2 1 3 Zlib006FcRn.Ihuman 25 5.5 5.5 4 1 4 Zlib006FcRn.I murine 100 7.4 7.4 2 1 5Zlib006FcRn.I murine 100 5.5 5.5 2 2 1-1 1 human 50 7.4 7.4 4 2 2-1 2human 50 7.4 5.5 4 2 2-2 2 human 25 5.5 7.4 6 2 3-1 3 human 5 5.5 7.4 42 3-2 3 human 5 5.5 5.5 8 2 4-1 4 murine 50 7.4 5.5 2 2 5-1 5 murine 1005.5 5.5 2 3 1-1-1 1-1 human 10 7.4 7.4 8 3 1-1-2 1-1 human 5 5.5 7.4 8 32-1-1 2-1 human 10 7.4 5.5 8 3 2-1-2 2-1 human 5 7.4 5.5 12 3 2-2-1 2-2human 10 7.4 5.5 12 3 2-2-2 2-2 human 5 7.4 5.5 15 3 3-1-1 3-1 human 15.5 7.4 8 3 3-2-1 3-2 human 0.5 5.5 5.5 12 3 3-2-2 3-2 human 0.25 5.55.5 16 3 4-1-1 4-1 murine 10 7.4 5.5 6 3 5-1-1 5-1 murine 5 5.5 5.5 8 41-1-1-1 1-1-1 human 1 7.4 7.4 12 4 1-1-1-2 1-1-1 human 0.25 7.4 7.4 15 41-1-2-1 1-1-2 human 0.5 7.4 5.5 15 4 1-1-2-2 1-1-2 human 0.1 5.5 7.4 154 2-1-1-1 2-1-1 human 1 7.4 5.5 15 4 2-1-1-2 2-1-1 human 0.5 7.4 5.5 154 2-1-2-1 2-1-2 human 0.25 7.4 5.5 20 (+IgG) 4 2-1-2-2 2-1-2 human 0.17.4 5.5 20 (+IgG) 4 2-2-1-1 2-2-1 and 2-2-2 human 0.5 5.5 7.4 15 42-2-2-1 2-2-1 and 2-2-2 human 0.5 7.4 5.5 20 4 3-1-1-1 3-1-1 human 1 5.57.4 12 4 3-2-1-1 3-2-1 and 3-2-2 human 0.5 5.5 5.5 16 4 4-1-1-1 4-1-1murine 1 7.4 5.5 12 4 5-1-1-1 5-1-1 murine 0.5 5.5 5.5 15

Amplification of Phew Particles:

Amplification of phage particles between selection cycle 1 and 2 wasperformed essentially as described in Example 2, with the followingexceptions. E. coli ER2738 was used for phage amplification and M13K07helper phage was used in 5× excess. The amplification of phage particlesbetween the selection cycles 2 and 4 was done by performing infection ofbacteria in solution as follows. After infection of log phase E. coliER2738 with phage particles, TSB supplemented with 2% glucose, 10 μg/mltetracycline and 100 μg/ml ampicillin was added, followed by incubationwith rotation for 30 min at 37° C. Thereafter, the bacteria wereinfected with M13K07 helper phage in 5× excess. The infected bacteriawere pelleted by centrifugation, re-suspended in TSB-YE mediumsupplemented with 100 μM IPTG, 25 μg/ml kanamycin and 100 μg/mlampicillin, and grown overnight at 30° C. The overnight cultures werepelleted in a centrifuge, and phage particles in the supernatant wereprecipitated twice with PEG/NaCl buffer. Finally, the phage particleswere re-suspended in selection buffer before entering the next selectioncycle.

In the final selection cycle, log phase bacteria were infected witheluate and diluted before spreading onto TBAB plates (30 g/l tryptoseblood agar base, Oxoid cat. no. CM0233B) supplemented with 0.2 g/lampicillin in order to form single colonies to be used in ELISAscreening.

Sequencing of Potential Binders:

Individual clones from the different selection tracks were picked forsequencing. All clones run in the ELISA screening were sequenced.Amplification of gene fragments and sequence analysis of gene fragmentswere performed essentially as described in Example 2.

ELISA Screening of Z Variants:

Single colonies containing Z variants (expressed as Z variant ABD fusionproteins as described in Example 2) were randomly picked from theselected clones of the FcRn maturated library and grown in 1 mlcultivations essentially as described in Example 2. Preparation of theperiplasmic supernatants was performed as in Example 2 with eight freezethawing cycles and the periplasmic fractions were used undiluted in theELISA screening. ELISA screenings were performed at both pH 6.0 and pH7.4 essentially as described in Example 2 using biotinylated human FcRnat a concentration of 2 nM in each well. The periplasmic fraction of theprimary FcRn binder Z10193 (SEQ ID NO:708; assayed in above experiments)was used as a positive control. Periplasm containing the ABD moiety onlywas used as a negative control.

ELISA K_(D) Analysis of FcRn Binding Z Variants:

A selection of FcRn binders was subjected to an analysis of the responseagainst a dilution series of biotinylated human FcRn using ELISA at bothpH 6.0 and pH 7.4 as described above. Biotinylated human FcRn was addedat a concentration of 30 nM and diluted stepwise 1:3 down to 14 μM. As abackground control, all Z variants were also assayed with no targetprotein added. Periplasm samples containing the primary FcRn binderZ07918 (SEQ ID. NO:707) was included and analyzed as a positive control.Periplasm containing the ABD moiety only was used as a negative control.Data were analyzed using Graph Pad Prism 5 and non-linear regression andK_(D) values (the half maximal effective concentration) were calculated.

Results

Phage Display Selection of Maturated FcRn Binding Z Variants:

Selection was performed in totally 14 parallel tracks containing fourcycles each. The different selection tracks differed in targetconcentration, target type (human FcRn or murine FcRn), selection time,and wash conditions.

Sequencing of Potential Binders:

Randomly picked clones were sequenced. Each individual Z variant wasgiven an identification number, Z #####, as described in Example 2. Intotal, 445 new unique Z variant molecules were identified.

The amino acid sequences of the 58 amino acid residues long Z variantsare listed in FIG. 1A-1MM and in the sequence listing as SEQ IDNO:723-1058. The deduced FcRn binding motifs of these Z variants arelisted in FIG. 1A-1MM and in the sequence listing as SEQ ID NO:17-352.The amino acid sequences of the 49 amino acid residues long polypeptidespredicted to constitute the complete three-helix bundle within each ofthese Z variants are listed in FIG. 1A-1MM and in the sequence listingas SEQ ID NO:370-705.

ELISA Screening of Z Variants:

Clones obtained after four selection cycles were produced in 96-wellplates and screened for FcRn binding activity using ELISA. All randomlypicked clones were analyzed. At pH 6.0, 333 of the 445 unique Z variantswere found to give a response of 0.3 AU or higher (corresponding to atleast 3× the negative control) against human FcRn at a concentration of2 nM. At pH 7.4, 278 of the 445 unique Z variants were found to give aresponse of 0.3 AU or higher (corresponding to at least 3× the negativecontrol) against human FcRn at a concentration of 2 nM. Clones with apositive signal against human FcRn were found in all tracks (includingthose with murine target) except 1-1-1-1. The negative controls hadabsorbances of 0.070-0.096 AU (pH 6.0) and 0.060-0.112 AU (pH 7.4),respectively. The average response of the blank controls was 0.070 AU(pH 6.0) and 0.062 (pH 7.4).

ELISA K_(D) Analysis of FcRn Binding Z Variants:

A subset of Z variants was selected based on the result in the ELISAexperiment described above (highest ELISA value at pH 6.0 and/or pH 7.4)and subjected to a target titration in ELISA format. Periplasm sampleswere incubated with a serial dilution of biotinylated human FcRn. Aperiplasm sample with the primary binder Z07918 (SEQ ID NO:707) was alsoassayed as a positive control. Obtained values were analyzed and theirrespective K_(D) values were calculated (Table 9).

TABLE 9 Calculated K_(D) values from ELISA titration analysis of Z-ABDvariants from the maturation. Z variant SEQ ID NO: K_(D) pH 6.0 (M)K_(D) pH 7.4 (M) Z13573 723 1.1 × 10⁻⁹ 3.8 × 10⁻⁹ Z13574 724 1.2 × 10⁻⁹5.0 × 10⁻⁹ Z13577 725  9.9 × 10⁻¹⁰ 1.4 × 10⁻⁹ Z13578 726 1.0 × 10⁻⁹ 2.5× 10⁻⁹ Z13579 727 1.2 × 10⁻⁹ 5.3 × 10⁻⁹ Z13581 728 1.1 × 10⁻⁹ 3.3 × 10⁻⁹Z13583 729  8.0 × 10⁻¹⁰ 1.5 × 10⁻⁹ Z13585 730 1.2 × 10⁻⁹ 1.7 × 10⁻⁹Z13586 731 1.2 × 10⁻⁹ 2.3 × 10⁻⁹ Z13587 732 1.4 × 10⁻⁹ 6.9 × 10⁻⁹ Z13588733 1.0 × 10⁻⁹ 2.3 × 10⁻⁹ Z13592 734  9.5 × 10⁻¹⁰ 1.8 × 10⁻⁹ Z13594 7351.3 × 10⁻⁹ 6.3 × 10⁻⁹ Z13596 736 1.5 × 10⁻⁹ 3.6 × 10⁻⁹ Z13597 737 1.4 ×10⁻⁹ 6.0 × 10⁻⁹ Z13598 738 1.1 × 10⁻⁹ 1.7 × 10⁻⁹ Z13600 739 1.4 × 10⁻⁹4.0 × 10⁻⁹ Z13604 740 1.3 × 10⁻⁹ 4.1 × 10⁻⁹ Z13605 741 1.3 × 10⁻⁹ 3.8 ×10⁻⁹ Z13609 742 1.3 × 10⁻⁹ 2.7 × 10⁻⁹ Z13611 743 1.3 × 10⁻⁹ 2.5 × 10⁻⁹Z13612 744 1.2 × 10⁻⁹ 8.6 × 10⁻⁹ Z13613 745 1.2 × 10⁻⁹ 4.3 × 10⁻⁹ Z13615746 1.2 × 10⁻⁹ 3.1 × 10⁻⁹ Z13616 747  9.6 × 10⁻¹⁰ 1.7 × 10⁻⁹ Z13617 7481.2 × 10⁻⁹ 1.9 × 10⁻⁹ Z13620 749 1.4 × 10⁻⁹ 3.3 × 10⁻⁹ Z13621 750  8.6 ×10⁻¹⁰ 1.4 × 10⁻⁹ Z13622 751 1.1 × 10⁻⁹ 2.1 × 10⁻⁹ Z13624 752 1.3 × 10⁻⁹3.4 × 10⁻⁹ Z13625 753 1.3 × 10⁻⁹ 2.8 × 10⁻⁹ Z13626 754 1.2 × 10⁻⁹ 2.7 ×10⁻⁹ Z13627 755 1.2 × 10⁻⁹ 2.9 × 10⁻⁹ Z13628 756 1.3 × 10⁻⁹ 5.5 × 10⁻⁹Z13629 757 1.2 × 10⁻⁹ 8.5 × 10⁻⁹ Z13633 758 1.5 × 10⁻⁹ 6.2 × 10⁻⁹ Z13634759 1.1 × 10⁻⁹ 2.3 × 10⁻⁹ Z13635 760 1.0 × 10⁻⁹ 1.7 × 10⁻⁹ Z13637 7611.3 × 10⁻⁹ 4.8 × 10⁻⁹ Z13638 762 1.2 × 10⁻⁹ 2.9 × 10⁻⁹ Z13639 763 1.3 ×10⁻⁹ 3.0 × 10⁻⁹ Z13640 764 1.1 × 10⁻⁹ 1.9 × 10⁻⁹ Z13641 765 1.1 × 10⁻⁹1.8 × 10⁻⁹ Z13644 766 1.3 × 10⁻⁹ 2.8 × 10⁻⁹ Z13645 767 1.2 × 10⁻⁹ 2.5 ×10⁻⁹ Z13648 768 1.6 × 10⁻⁹ 3.3 × 10⁻⁹ Z13651 769 1.2 × 10⁻⁹ 2.7 × 10⁻⁹Z13652 770 1.4 × 10⁻⁹ 2.9 × 10⁻⁹ Z13654 771  9.5 × 10⁻¹⁰ 2.9 × 10⁻⁹Z13655 772 1.1 × 10⁻⁹ 2.4 × 10⁻⁹ Z13656 773 1.1 × 10⁻⁹ 3.7 × 10⁻⁹ Z13657774 2.1 × 10⁻⁹ 3.9 × 10⁻⁹ Z13659 775 2.2 × 10⁻⁹ 3.1 × 10⁻⁹ Z13663 776 9.3 × 10⁻¹⁰ 1.5 × 10⁻⁹ Z13664 777 2.4 × 10⁻⁹ 4.2 × 10⁻⁹ Z13667 778 1.2× 10⁻⁹ 2.3 × 10⁻⁹ Z13669 779  9.2 × 10⁻¹⁰ 1.7 × 10⁻⁹ Z13672 780 2.5 ×10⁻⁹ 5.6 × 10⁻⁹ Z13674 781  9.2 × 10⁻¹⁰ 1.3 × 10⁻⁹ Z13675 782  9.6 ×10⁻¹⁰ 2.2 × 10⁻⁹ Z13676 783  9.4 × 10⁻¹⁰ 3.1 × 10⁻⁹ Z13678 784 2.0 ×10⁻⁹ 3.3 × 10⁻⁹ Z13684 785 1.0 × 10⁻⁹ 2.2 × 10⁻⁹ Z13688 786 1.3 × 10⁻⁹2.1 × 10⁻⁹ Z13691 787 1.8 × 10⁻⁹ 2.7 × 10⁻⁹ Z13692 788 1.3 × 10⁻⁹ 3.7 ×10⁻⁹ Z13694 789  9.8 × 10⁻¹⁰ 3.6 × 10⁻⁹ Z13695 790 1.8 × 10⁻⁹ 5.3 × 10⁻⁹Z13697 791 1.2 × 10⁻⁹ 2.4 × 10⁻⁹ Z13706 792 2.0 × 10⁻⁹ 6.4 × 10⁻⁹ Z13708793 1.9 × 10⁻⁹ 4.4 × 10⁻⁹ Z13710 794 1.6 × 10⁻⁹ 2.6 × 10⁻⁹ Z13711 7952.1 × 10⁻⁹ 4.9 × 10⁻⁹ Z13714 796 2.1 × 10⁻⁹ 6.0 × 10⁻⁹ Z13716 797 1.8 ×10⁻⁹ 5.8 × 10⁻⁹ Z13719 798 2.6 × 10⁻⁹ 7.3 × 10⁻⁹ Z13720 799 2.5 × 10⁻⁹4.5 × 10⁻⁷ Z13721 800 1.9 × 10⁻⁹ 2.9 × 10⁻⁹ Z13725 801 1.8 × 10⁻⁹ 4.9 ×10⁻⁹ Z13727 802 2.1 × 10⁻⁹ 5.9 × 10⁻⁹ Z13728 803 2.6 × 10⁻⁹ 6.7 × 10⁻⁹Z13732 804 2.1 × 10⁻⁹ 9.4 × 10⁻⁹ Z13735 805 1.6 × 10⁻⁹ 9.1 × 10⁻⁹ Z13736806 1.7 × 10⁻⁹ 3.0 × 10⁻⁹ Z13740 807 2.0 × 10⁻⁹ 5.0 × 10⁻⁹ Z13742 8082.4 × 10⁻⁹ 7.6 × 10⁻⁹ Z13747 809 1.3 × 10⁻⁹ 2.3 × 10⁻⁹ Z13749 810 2.8 ×10⁻⁹ 1.2 × 10⁻⁸ Z13750 811 2.7 × 10⁻⁹ 8.4 × 10⁻⁹ Z13751 812 2.0 × 10⁻⁹3.8 × 10⁻⁹ Z13752 813 2.0 × 10⁻⁹ 5.8 × 10⁻⁹ Z13758 814 1.9 × 10⁻⁹ 6.5 ×10⁻⁹ Z13759 815 2.1 × 10⁻⁹ 5.6 × 10⁻⁹ Z13760 816 2.1 × 10⁻⁹ 5.8 × 10⁻⁹Z13761 817 1.9 × 10⁻⁹ 3.7 × 10⁻⁹ Z13771 818 1.5 × 10⁻⁹ 2.0 × 10⁻⁹ Z13773819 2.5 × 10⁻⁹ 4.9 × 10⁻⁹ Z13776 820 2.2 × 10⁻⁹ 5.5 × 10⁻⁹ Z13777 8212.4 × 10⁻⁹ 4.6 × 10⁻⁹ Z13780 822 2.1 × 10⁻⁹ 4.0 × 10⁻⁹ Z13782 823 2.2 ×10⁻⁹ 4.2 × 10⁻⁹ Z13783 824 1.4 × 10⁻⁹ 2.2 × 10⁻⁹ Z13786 825 2.3 × 10⁻⁹4.7 × 10⁻⁹ Z13792 826 2.0 × 10⁻⁹ 2.9 × 10⁻⁹ Z13796 827 2.3 × 10⁻⁹ 4.2 ×10⁻⁹ Z13799 828 1.9 × 10⁻⁹ 5.6 × 10⁻⁹ Z13806 829 1.6 × 10⁻⁹ 3.1 × 10⁻⁹Z13808 830 2.4 × 10⁻⁹ 5.5 × 10⁻⁹ Z13811 831 2.0 × 10⁻⁹ 3.1 × 10⁻⁹ Z13812832 2.3 × 10⁻⁹ 1.1 × 10⁻⁸ Z13823 833 2.9 × 10⁻⁹ 3.8 × 10⁻⁹ Z13824 8341.9 × 10⁻⁹ 3.8 × 10⁻⁹ Z13838 835 2.6 × 10⁻⁹ 5.4 × 10⁻⁹ Z13840 836 2.2 ×10⁻⁹ 4.1 × 10⁻⁹ Z13842 837 2.2 × 10⁻⁹ 5.5 × 10⁻⁹ Z13845 838 2.6 × 10⁻⁹4.2 × 10⁻⁹ Z13846 839 2.3 × 10⁻⁹ 4.3 × 10⁻⁹ Z13848 840 2.1 × 10⁻⁹ 3.1 ×10⁻⁹ Z13849 841 2.1 × 10⁻⁹ 3.0 × 10⁻⁹ Z13860 842 2.3 × 10⁻⁹ 8.7 × 10⁻⁹Z13865 843 2.5 × 10⁻⁹ 5.6 × 10⁻⁹ Z13866 844 2.0 × 10⁻⁹ 2.8 × 10⁻⁹ Z13875845 2.0 × 10⁻⁹ 3.4 × 10⁻⁹ Z13879 846 2.1 × 10⁻⁹ 3.0 × 10⁻⁹

Example 9 Production and Characterization of Z Variants from a MaturatedLibrary

In this Example, twelve Z variants were produced in E. coli, purifiedand assayed for binding to FcRn as well as for inhibition of IgG bindingto FcRn.

Materials and Methods

Subcloning of Z variants into expression vectors: The DNA of twelve FcRnbinding Z variants (Z13577 (SEQ ID NO:725), Z13578 (SEQ ID NO:726),Z13583 (SEQ ID NO:729), Z13592 (SEQ ID NO:734), Z13616 (SEQ ID NO:747),Z13621 (SEQ ID NO:750), Z13654 (SEQ ID NO:771), Z13663 (SEQ ID NO:776),Z13669 (SEQ ID NO:779), Z13674 (SEQ ID NO:781), Z13675 (SEQ ID NO:782)and Z13676 (SEQ ID NO:783)) were amplified from the library vectorpAY02592. The subcloning was performed as described in Example 3. The Zgene fragments were subcloned into the expression vector pAY01448resulting in the encoded sequence MGSSHHHHHHLQ-[Z #####]-VD (SEQ ID NO:1106).

Production of Z Variants:

Cultivation and purification of the His₆-tagged Z variants was performedessentially as described in Example 3.

Biacore Binding and Kinetic Analyses:

The interaction of FcRn binding His₆-tagged Z variants with human FcRnwas analyzed in a Biacore 2000 instrument essentially as described inExample 3. Human FcRn purchased from Biorbyt (cat. no. orb84388) wasused as target protein. The analytes were injected during 2 minutes at30 μl/min. The dissociation phase was 4 minutes and the equilibrationtime between the analyte injections was 30 minutes.

In one experiment, the Z variants were injected at pH 6.0 followed bydissociation in buffers of pH 6.0 or pH 7.4, respectively, using theco-inject procedure. The concentration of the Z variants was 100 nM.

In another experiment, approximate kinetic constants (k_(on) andk_(off)) and affinities (K_(D)) were determined for a subset of Zvariants. Injected concentrations were 540 nM, 180 nM, 60 nM, 20 nM and6.7 nM.

AlphaLISA Blocking Assay:

The potential of Z variants to inhibit binding of IgG to FcRn wasanalyzed in the AlphaLISA assay described in Example 3.

Results

Production of Z Variants:

The twelve FcRn binding Z variants constructed with an N-terminal His₆tag were produced in E. coli. SDS-PAGE analysis of each final proteinpreparation showed that these predominantly contained the FcRn binding Zvariant. The correct identity and molecular weight of each FcRn bindingZ variant was confirmed by HPLC-MS analysis.

Biacore Binding and Kinetic Analyses:

The binding of the twelve Z variants to human FcRn and the dissociationat different pH were tested in a Biacore instrument by sequentiallyinjecting each of the Z variants at pH 6.0 and either buffer pH 6.0 orpH 7.4 over a chip surface containing FcRn. The ligand immobilizationlevel of the surface was 890 RU human FcRn. The twelve Z variants showedbinding to FcRn at pH 6.0, and for all variants, faster off-rates wereseen at pH 7.4 compared to pH 6.0.

The kinetic constants of the Z variants Z13577 (SEQ ID NO:725) andZ13621 (SEQ ID NO:750) interacting with FcRn at pH 6.0 were determined(see Table 10). Kinetic constants were calculated using curve sets oftwo or four injected concentrations of Z13577 and Z13621, respectively.

TABLE 10 Biacore kinetic constants and affinities for FcRn binding at pH6.0. Z variant SEQ ID NO: k_(on) (M⁻¹s−1) k_(off) (s⁻¹) K_(D) (M) Z13577725 3.0 × 10⁵ 4.0 × 10⁻³ 13 × 10⁻⁹  Z13621 750 6.4 × 10⁵ 3.7 × 10⁻³ 6 ×10⁻⁹

AlphaLISA Blocking Analysis:

The ability of twelve maturated His₆-tagged monomeric Z variants toinhibit IgG binding to FcRn was tested in an AlphaLISA blocking assay.Serial dilutions of the Z variants were incubated with biotinylatedhuman FcRn and the blocking ability of each respective variant wasmeasured after addition of IgG coated Acceptor beads and subsequentlystreptavidin coated Donor beads. Inhibition could be measured as adecrease in AlphaLISA counts for positive Z variants. All twelve testedZ variants were shown to block IgG binding to FcRn and the calculatedIC50 values are shown in Table 11.

TABLE 11 Calculated IC50 values from AlphaLISA blocking assay. Z variantSEQ ID NO: IC50 (M) Z13577 725 1.2 × 10⁸ Z13578 726 1.2 × 10⁸ Z13583 7292.7 × 10⁹ Z13592 734 6.4 × 10⁹ Z13616 747 7.4 × 10⁹ Z13621 750 3.2 × 10⁹Z13654 771 3.5 × 10⁹ Z13663 776 1.1 × 10⁸ Z13669 779 5.2 × 10⁹ Z13674781 2.5 × 10⁹ Z13675 782 8.2 × 10⁹ Z13676 783 3.9 × 10⁹

Example 10 Comparison of Blocking Capacity of IgG Binding to FcRn

In this example, the IgG blocking capacity of the FcRn binding Z variantHis₆-Z07918 (SEQ ID NO:707) was compared to Intravenous immunoglobulin(IVIg) and Subcutaneous immunoglobulin (SCIg) currently used in thetreatment of some autoimmune disorders.

Materials and Methods

Blocking of IgG-FcRn immunofluorescence staining: Human or murineFcRn-eGFP transduced HeLa cells were prepared as described in Example 4.Fixed cells were resuspended in 50 μl of a mix of 50 nMAlexa647-conjugated human IgG (Jackson laboratories, cat. no.009-600-003) and His₆-tagged Z07918, IVIg (OCTAGRAM, Octapharma) or SCIg(GAMMANORM, Octapharma), respectively, diluted at concentrations of1000, 100, 10, 1, 0.1 or 0 (buffer control) nM in Mcllvanes, pH 6.0,plus 2.5% FCS (Ultra low IgG, Life technologies) and 0.1% saponin(AppliChem). The cells were incubated for 1 h at 37° C. in the dark,washed with 2×100 μl Mcllvanes, pH 6.0, plus 2.5% FCS (Ultra low IgG) pH6.0 and re-suspended in 180 μl of Mcllvanes, pH 6.0, plus 1% BSA. Datafrom 10,000 GFP/FcRn positive cells were obtained using a FACS Calibur(Beckman Coulter) and the data was analyzed using Flowing software 2.5.0(Turku University).

Results

The experiment was performed to determine if the FcRn binding Z variantHis₆-Z07918 (SEQ ID NO:707) blocks the IgG-FcRn interaction and comparethe blocking effect to IVIg and SCIg. Human or murine FcRn-eGFPtransduced HeLa cells were incubated with human Alexa647-conjugated IgG.The binding was blocked with unlabeled His₆-Z07918, IVIg or SCIg atdifferent concentrations. The results showed that His₆-Z07918effectively blocked hIgG binding to hFcRn to a similar extent as IVIg orSCIg (FIG. 9).

Example 11 Increased IgG Catabolism by FcRn Binding Z Variants in Mice

The ability of the FcRn binding Z variant Z07918 to block IgG binding toFcRn in vitro was shown in Example 10. In this example, the blockingability of the same Z variant was evaluated in vivo. Blocking ofIgG-FcRn interactions in vivo will lead to increased IgG catabolism andconcomitant reduced levels of IgG (Mezo 2008, supra).

Materials and Methods

Animal Study:

The FcRn-binding Z variants Z11948 (SEQ ID NO:1060) and Z07918-PP013(Z07918 (SEQ ID NO:707) identical to Z11948 but with the N-terminusstarting with the amino acids VD instead of AE, in fusion with the ABDvariant PP013 (SEQ ID NO:1063)) or vehicle (PBS buffer), wereadministered to male NMRI (Charles River), at a dose of 16.3 μmol/kg.The mice were treated with five intravenous injections given at 0, 24,48, 72 and 96 h. Serum samples were taken at 0, 72, 120 and 168 h(termination of study) and stored at −20° C. The concentration of mouseIgG in serum was quantified by ELISA.

Mouse IgG ELISA:

The concentration of mouse IgG in mouse serum samples was analyzed by amouse IgG ELISA kit (Mabtech 3825-1AD-6) and performed as described bythe manufacturer. The concentration of mIgG was calculated from astandard curve provided and GraphPad prism5 using a non-linearregression formula. The concentration of IgG in individual mice at 24,72, 120 and 168 h were related to the level at 0 h and the results aretherefore presented as percentage of IgG (0 h).

Results

The results showed a reduction of mouse IgG concentration in micetreated with FcRn-specific Z variants. Both Z11948 and the ABD-fusedvariant Z07918-PP013 lowered the concentration of endogenous IgG in micein vivo. Most pronounced effects were obtained with the ABD-fusedvariant and after 120 hours. Thus, the results indicates that theFcRn-specific Z variants blocked recycling of IgG resulting in increasedIgG catabolism and subsequent lower levels of IgG in mice.

Example 12 In Vitro Transcytosis of FcRn Binding Z Variants

In this example, the FcRn binding Z variants are tested for theirability to be transported through epithelial or endothelial cells orrecycled by FcRn in vitro. A drug containing a Z variant with the powerof transcytosis will facilitate drug uptake after for example oral orpulmonary administration.

Materials and Methods

Cells, for example T84, MDCK, HeLa, CaCo2, CaLu-1 and/or CaLu-3 cells,with or without endogenous or recombinant expression of FcRn, are grownin respective growth medium on a membrane in a transwell to form amonolayer. The integrity of monolayers can be evaluated by measuring theelectrical resistance or adding a probe that is not able to penetrate orbeing actively transported over the cell monolayer. A defined monolayerof cells is pulsed from the apical or basolateral side with ligand suchas FcRn binding Z variants, HSA or IgG in a buffer such as HBSS (Hanks'Balanced Salt Solution, SigmaAldrich, cat. no. H9269) or growth mediumat a suitable pH and temperature, and chased with buffers such as HBSSor growth medium at a suitable pH and temperature on the opposite side.

In a variant of this assay, ligands can be chased with buffers such asHBSS or growth medium at suitable pH and temperature on the same side asadministration to measure recycled ligand as well. This can be done in atranswell or in a cell culture dish. Cells are seeded into transwell orcell culture dishes and pulsed with ligands such as FcRn binding Zvariants, HSA or IgG. Endocytosed ligands will bind to FcRn and returnto the cell surface at the same or opposite side as they were loaded.After pulsing, free ligands are removed by washing the cells with coldbuffer. To chase ligands, warm buffer or medium is added to the cellsand, after a period in the range from 10 minutes to several hours, thebuffer or medium is removed and assayed for the presence of ligands.

In a variant of this assay, ligands such as FcRn binding Z variants, HSAor IgG can be used to block the binding to FcRn by ligands such as otherFcRn binding Z variants, HSA or IgG by administering them at the sametime or sequentially to the cells.

The amount of ligand can be quantified by methods such as ELISA,HPLC-MS, fluorescent dye or radio labeling.

The results of the experiment described above are expected to show thatthe FcRn-specific Z variants can be transcytosed and/or recycled invitro.

Itemized Listing of Embodiments

1. FcRn binding polypeptide, comprising an FcRn binding motif BM, whichmotif consists of the amino acid sequence

(SEQ ID NO: 1075) EX₂ X₃ X₄ AX₆ X₇ EIR WLPNLX₁₆X₁₇ X₁₈QR X₂₁ AFIX₂₅ X₂₆LX₂₈ X₂₉wherein, independently from each other,

X₂ is selected from A, D, E, F, H, I, K, L, N, Q, R, S, T, V, W and Y;

X₃ is selected from A, D, E, F, G, H, I, K, L, M, N, Q, R, S, T, V, Wand Y;

X₄ is selected from A, D, E, F, G, H, I, K, L, N, Q, R, S, T, V, W andY;

X₆ is selected from A, E, F, G, H, I, K, Q, R, S and V;

X₇ is selected from A, F, H, K, N, Q, R, S and V;

X₁₆ is selected from N and T;

X₁₇ is selected from F, W and Y;

X₁₈ is selected from A, D, E and N;

X₂₁ is selected from A, S, V and W;

X₂₅ is selected from D, E, G, H, I, K, L, N, Q, R, S, T, V, W and Y;

X₂₆ is selected from K and S,

X₂₈ is selected from A, D, E, F, H, I, K, L, N, Q, R, S, T, V, W and Y;and

X₂₉ is selected from D and R.

2. FcRn binding polypeptide according to item 1, wherein, independentlyfrom each other,

X₂ is selected from A, D, E, F, H, I, K, L, N, Q, R, S, T, V, W and Y;

X₃ is selected from A, D, E, F, H, I, K, L, M, N, Q, R, S, T, V, W andY;

X₄ is selected from A, D, E, F, H, I, K, L, N, Q, R, S, T, V, W and Y;

X₆ is selected from A, E, F, G, H, I, K, Q, R and S,

X₇ is selected from A, F, H, K, N, Q, R, S and V;

X₁₆ is selected from N and T;

X₁₇ is selected from F and Y;

X₁₈ is D;

X₂₁ is V;

X₂₅ is selected from D, E, H, I, K, L, N, Q, R, S, T, V, W and Y;

X₂₆ is selected from K and S,

X₂₈ is selected from A, D, E, F, H, I, K, L, N, Q, R, S, T, V and W; and

X₂₉ is selected from D and R.

3. FcRn binding polypeptide according to item 1, wherein the BM consistsof an amino acid sequence selected from

i) (SEQ ID NO: 1076) EX₂ X₃ X₄ AX₆ HEIR WLPNLTX₁₇ X₁₈ QRX₂₁ AFIX₂₅ KLX₂₈ Dwherein, independently from each other,

X₂ is selected from A, D, E, F, H, I, K, L, N, Q, R, S, T, V, W and Y;

X₃ is selected from A, D, E, G, H, K, L, M, N, Q, R, S, T, V and Y;

X₄ is selected from A, D, E, F, G, I, K, L, N, Q, R, S, T, V and Y;

X₆ is selected from A, G, K, R, S and V;

X₁₇ is selected from F, W and Y;

X₁₈ is selected from A, D, E and N;

X₂₁ is selected from A, S, V and W;

X₂₈ is selected from D, G, H, K, L, N, R, V and W;

X₂₈ is selected from A, D, E, H, K, L, N, Q, R, S, T, W and Y; and

-   -   ii) an amino acid sequence which has at least 96% identity to a        sequence defined by i).        4. FcRn binding polypeptide according to any preceding item,        wherein X₂ is selected from A, D, E, F, I, L, N, Q, R, S, T, V,        W and Y.        5. FcRn binding polypeptide according to item 4, wherein X₂ is        selected from A, D, F, I, L, N, Q, R, S, T, V, W and Y.        6. FcRn binding polypeptide according to item 5, wherein X₂ is        selected from A, D, F, I, L, N, Q, R, S, V and W.        7. FcRn binding polypeptide according to item 5, wherein X₂ is        selected from A, I, L, N, Q, R, S, T, V, W and Y.        8. FcRn binding polypeptide according to item 7, wherein X₂ is        selected from A, I, L, N, Q, S, T, V and W.        9. FcRn binding polypeptide according to item 6 or 8, wherein X₂        is selected from A, I, L, N, Q, V and W.        10. FcRn binding polypeptide according to item 9, wherein X₂ is        selected from A, I, L, Q, V and W.        11. FcRn binding polypeptide according to item 10, wherein X₂ is        selected from A, I, L and Q.        12. FcRn binding polypeptide according to item 11, wherein X₂ is        selected from I, L and Q.        13. FcRn binding polypeptide according to item 12, wherein X₂ is        selected from I and Q.        14. FcRn binding polypeptide according to item 13, wherein X₂ is        I.        15. FcRn binding polypeptide according to item 13, wherein X₂ is        Q.        16. FcRn binding polypeptide according to any one of items 1 and        3-15, wherein X₃ is selected from A, D, E, G, H, K, L, M, N, Q,        R, S, T, V and Y.        17. FcRn binding polypeptide according to item 2 or 16, wherein        X₃ is selected from A, D, E, H, K, L, M, N, Q, R, S, T, V and Y.        18. FcRn binding polypeptide according to item 16, wherein X₃ is        selected from A, D, E, G, H, K, L, M, N, Q, R, S and T.        19. FcRn binding polypeptide according to item 18, wherein X₃ is        selected from A, D, E, G, H, K, M, N, Q, S and T.        20. FcRn binding polypeptide according to item 19, wherein X₃ is        selected from A, D, E, G, H, M, N, Q, S and T.        21. FcRn binding polypeptide according to item 19, wherein X₃ is        selected from A, D, E, K, N, Q, S and T.        22. FcRn binding polypeptide according to item 21, wherein X₃ is        selected from A, D, E, K, Q and T.        23. FcRn binding polypeptide according to item 22, wherein X₃ is        selected from A, D, E, Q and T.        24. FcRn binding polypeptide according to item 23, wherein X₃ is        selected from D, E and T.        25. FcRn binding polypeptide according to item 24, wherein X₃ is        selected from D and E.        26. FcRn binding polypeptide according to item 25, wherein X₃ is        D.        27. FcRn binding polypeptide according to item 25, wherein X₃ is        E.        28. FcRn binding polypeptide according to any one of items 1 and        3-27, wherein X₄ is selected from A, D, E, F, G, I, K, L, N, Q,        R, S, T, V and Y.        29. FcRn binding polypeptide according to item 28, wherein X₄ is        selected from A, D, E, G, N, Q, R, S, T and V.        30. FcRn binding polypeptide according to item 2 or 28, wherein        X₄ is selected from A, D, E, F, I, K, L, N, Q, R, S, T and V.        31. FcRn binding polypeptide according to item 30, wherein X₄ is        selected from A, D, E, I, K, N, Q, R, S and T.        32. FcRn binding polypeptide according to item 31, wherein X₄ is        selected from A, D, E, I, K, Q, S and T.        33. FcRn binding polypeptide according to item 32, wherein X₄ is        selected from A, D, I, K, Q and S.        34. FcRn binding polypeptide according to item 32, wherein X₄ is        selected from A, D, E, K and S.        35. FcRn binding polypeptide according to item 33 or 34, wherein        X₄ is selected from A, D, K and S.        36. FcRn binding polypeptide according to item 34, wherein X₄ is        selected from A, D, E and K.        37. FcRn binding polypeptide according to item 35 or 36, wherein        X₄ is selected from A, D and K.        38. FcRn binding polypeptide according to item 37, wherein X₄ is        selected from A and D.        39. FcRn binding polypeptide according to item 36, wherein X₄ is        selected from A and E.        40. FcRn binding polypeptide according to item 38 or 39, wherein        X₄ is A.        41. FcRn binding polypeptide according to item 38, wherein X₄ is        D.        42. FcRn binding polypeptide according to item 39, wherein X₄ is        E.        43. FcRn binding polypeptide according to any one of items 1 and        4-42, wherein X₆ is selected from A, G, K, Q, R, S and V.        44. FcRn binding polypeptide according to item 3 or 43, wherein        X₆ is selected from A, G, K, R, S and V.        45. FcRn binding polypeptide according to item 2 or 44, wherein        X₆ is selected from A, G, K, R and S.        46. FcRn binding polypeptide according to item 44, wherein X₆ is        selected from A, G, K, S and V.        47. FcRn binding polypeptide according to item 46, wherein X₆ is        selected from A, G, K and V.        48. FcRn binding polypeptide according to item 45 or 46, wherein        X₆ is selected from A, G, K and S.        49. FcRn binding polypeptide according to item 47 or 48, wherein        X₆ is selected from A, G and K.        50. FcRn binding polypeptide according to item 47, wherein X₆ is        selected from A, G and V.        51. FcRn binding polypeptide according to item 49 or 50, wherein        X₆ is selected from A and G.        52. FcRn binding polypeptide according to item 51, wherein X₆ is        A.        53. FcRn binding polypeptide according to item 51, wherein X₆ is        G.        54. FcRn binding polypeptide according to any one of items 1, 2        and 4-53, wherein X₇ is selected from A and H.        55. FcRn binding polypeptide according to item 54, wherein X₇ is        H.        56. FcRn binding polypeptide according to any one of items 1, 2        and 4-55, wherein X₁₆ is T.        57. FcRn binding polypeptide according to any one of items 1, 2        and 4-55, wherein X₁₆ is N.        58. FcRn binding polypeptide according to any one of items 1 and        3-57, wherein X₁₇ is selected from F and Y.        59. FcRn binding polypeptide according to any preceding item,        wherein X₁₇ is F.        60. FcRn binding polypeptide according to any one of items 1 and        3-59, wherein X₁₈ is selected from A, D and E.        61. FcRn binding polypeptide according to item 60, wherein X₁₈        is selected from A and D.        62. FcRn binding polypeptide according to item 61, wherein X₁₈        is D.        63. FcRn binding polypeptide according to any one of items 1 and        3-62, wherein X₂₁ is selected from V and W.        64. FcRn binding polypeptide according to item 63, wherein X₂₁        is V.        65. FcRn binding polypeptide according to any one of items 1 and        4-64, wherein X₂₅ is selected from D, E, G, H, K, L, N, Q, R, V        and W.        66. FcRn binding polypeptide according to item 65, wherein X₂₅        is selected from D, G, H, K, L, N, R, V and W.        67. FcRn binding polypeptide according to any one of items 2, 3        and 66, wherein X₂₅ is selected from H, L, R, V and W.        68. FcRn binding polypeptide according to item 67, wherein X₂₅        is selected from H, R, V and W.        69. FcRn binding polypeptide according to item 68, wherein X₂₅        is selected from H, R and V.        70. FcRn binding polypeptide according to item 67, wherein X₂₅        is selected from H, L and R.        71. FcRn binding polypeptide according to item 69 or 70, wherein        X₂₅ is selected from H and R.        72. FcRn binding polypeptide according to item 69, wherein X₂₅        is selected from H and V.        73. FcRn binding polypeptide according to item 71 or 72, wherein        X₂₅ is H.        74. FcRn binding polypeptide according to any one of items 1, 2        and 4-73, wherein X₂₆ is K.        75. FcRn binding polypeptide according to any one of items 1, 2        and 4-73, wherein X₂₆ is S.        76. FcRn binding polypeptide according to any one of items 1 and        3-75, wherein X₂₈ is selected from A, D, E, H, K, L, N, Q, R, S,        T, W and Y.        77. FcRn binding polypeptide according to item 76, wherein X₂₈        is selected from A, D, E, K, L, N, Q, R, S, T, W and Y.        78. FcRn binding polypeptide according to item 77, wherein X₂₈        is selected from A, D, E, L, R, S, T, W and Y.        79. FcRn binding polypeptide according to item 2 or 77, wherein        X₂₈ is selected from A, D, K, L, N, Q, R, S, T and W.        80. FcRn binding polypeptide according to item 78 or 79, wherein        X₂₈ is selected from A, D and R.        81. FcRn binding polypeptide according to item 80, wherein X₂₈        is selected from A and R.        82. FcRn binding polypeptide according to item 80, wherein X₂₈        is selected from D and R.        83. FcRn binding polypeptide according to item 81, wherein X₂₈        is A.        84. FcRn binding polypeptide according to item 81 or 82, wherein        X₂₈ is R.        85. FcRn binding polypeptide according to item 82, wherein X₂₈        is D.        86. FcRn binding polypeptide according to any one of items 1, 2        and 4-85, wherein X₂₉ is D.        87. FcRn binding polypeptide according to any one of items 1, 2        and 4-85, wherein X₂₉ is R.        88. FcRn binding polypeptide according to any one of items 1, 2        and 4-87, wherein X₆X₇ is selected from AH and GH.        89. FcRn binding polypeptide according to item 88, wherein X₆X₇        is AH.        90. FcRn binding polypeptide according to item 88, wherein X₆X₇        is GH.        91. FcRn binding polypeptide according to any preceding item,        wherein X₁₇X₁₈ is selected from FD and YD.        92. FcRn binding polypeptide according to item 91, wherein        X₁₇X₁₈ is FD.        93. FcRn binding polypeptide according to any preceding item,        wherein the sequence fulfills at least three of the six        conditions I-VI:    -   I. X₆ is selected from A, G, K and S, such as in particular A;    -   II. X₇ is H,    -   III. X₁₇ is selected from F and Y, such as in particular F;    -   IV. X₁₈ is D;    -   V. X₂₁ is selected from V and W, such as in particular V;    -   VI. X₂₅ is selected from H and R, such as in particular H.        94. FcRn binding polypeptide according to item 93, wherein the        sequence fulfills at least four of the six conditions I-VI.        95. FcRn binding polypeptide according to item 94, wherein the        sequence fulfills at least five of the six conditions I-VI.        96. FcRn binding polypeptide according to item 95, wherein the        sequence fulfills all of the six conditions I-VI.        97. FcRn binding polypeptide according to any preceding item,        wherein the sequence is selected from the group consisting of        SEQ ID NO:1-353.        98. FcRn binding polypeptide according to item 97, wherein the        sequence is selected from the group consisting of SEQ ID        NO:1-15, SEQ ID NO:17-140 and SEQ ID NO:353.        99. FcRn binding polypeptide according to item 98, wherein the        sequence is selected from the group consisting of SEQ ID NO:1-2        and SEQ ID NO:17-140.        100. FcRn binding polypeptide according to item 99, wherein the        sequence is selected from the group consisting of SEQ ID NO:1-2,        SEQ ID NO:17-92, SEQ ID NO:94-103, SEQ ID NO:105-125 and SEQ ID        NO:127-140.        101. FcRn binding polypeptide according to item 98, wherein the        sequence is selected from the group consisting of SEQ ID NO:1-8,        SEQ ID NO:13, SEQ ID NO:19-20, SEQ ID NO:23, SEQ ID NO:28, SEQ        ID NO:41, SEQ ID NO:44, SEQ ID NO:65, SEQ ID NO:70, SEQ ID        NO:73, SEQ ID NO:75-77 and SEQ ID NO:353.        102. FcRn binding polypeptide according to item 100 or 101,        wherein the sequence is selected from the group consisting of        SEQ ID NO:1, SEQ ID NO:23, SEQ ID NO:28, SEQ ID NO:41, SEQ ID        NO:44, SEQ ID NO:65, SEQ ID NO:73 and SEQ ID NO:75-77.        103. FcRn binding polypeptide according to item 102, wherein the        sequence is selected from the group consisting of SEQ ID NO:1,        SEQ ID NO:23, SEQ ID NO:44, SEQ ID NO:65, SEQ ID NO:75 and SEQ        ID NO:77.        104. FcRn binding polypeptide according to item 103, wherein the        sequence is selected from the group consisting of SEQ ID NO:1,        SEQ ID NO:23 and SEQ ID NO:75.        105. FcRn binding polypeptide according to item 104, wherein the        sequence is SEQ ID NO:1.        106. FcRn binding polypeptide according to any preceding item,        wherein said FcRn binding motif forms part of a three-helix        bundle protein domain.        107. FcRn binding polypeptide according to item 106, wherein        said FcRn binding motif essentially forms part of two helices        with an interconnecting loop, within said three-helix bundle        protein domain.        108. FcRn binding polypeptide according to item 107, wherein        said three-helix bundle protein domain is selected from        bacterial receptor domains.        109. FcRn binding polypeptide according to item 108, wherein        said three-helix bundle protein domain is selected from domains        of protein A from Staphylococcus aureus or derivatives thereof.        110. FcRn binding polypeptide according to any preceding item,        which comprises an amino acid sequence selected from:    -   iii) K-[BM]-DPSQS X_(a)X_(b)LLX_(c) EAKKL X_(d)X_(e)X_(f)Q (SEQ        ID NO: 1077);        wherein    -   [BM] is an FcRn binding motif as defined herein, provided that        X₂₉ is D;    -   X_(a) is selected from A and S,    -   X_(b) is selected from N and E;    -   X_(c) is selected from A, S and C;    -   X_(d) is selected from E, N and S,    -   X_(e) is selected from D, E and S,    -   X_(f) is selected from A and S, and    -   iv) an amino acid sequence which has at least 93% identity to a        sequence defined by iii).        111. FcRn binding polypeptide according to any one of items        1-109, which comprises an amino acid sequence selected from

v) (SEQ ID NO: 1080)K-[BM]-QPEQS X_(a)X_(b)LLX_(c) EAKKL X_(d)X_(e)X_(f)Q;wherein

-   -   [BM] is an FcRn binding motif as defined herein, provided that        X₂₉ is R;    -   X_(a) is selected from A and S,    -   X_(b) is selected from N and E;    -   X_(c) is selected from A, S and C;    -   X_(d) is selected from E, N and S,    -   X_(e) is selected from D, E and S,    -   X_(f) is selected from A and S, and    -   vi) an amino acid sequence which has at least 93% identity to a        sequence defined by v).        112. FcRn binding polypeptide according to item 110 or 111,        wherein X_(a) in sequence iii) or v) is A.        113. FcRn binding polypeptide according to item 110 or 111,        wherein X_(a) in sequence iii) or v) is S.        114. FcRn binding polypeptide according to any one of items        110-113, wherein X_(b) in sequence iii) or v) is N.        115. FcRn binding polypeptide according to any one of items        110-113, wherein X_(b) in sequence iii) or v) is E.        116. FcRn binding polypeptide according to any one of items        110-115, wherein X_(c) in sequence iii) or v) is A.        117. FcRn binding polypeptide according to any one of items        110-115, wherein X_(c) in sequence iii) or v) is S.        118. FcRn binding polypeptide according to any one of items        110-115, wherein X_(c) in sequence iii) or v) is C.        119. FcRn binding polypeptide according to any one of items        110-118, wherein X_(d) in sequence iii) or v) is E.        120. FcRn binding polypeptide according to any one of items        110-118, wherein X_(d) in sequence iii) or v) is N.        121. FcRn binding polypeptide according to any one of items        110-118, wherein X_(d) in sequence iii) or v) is S.        122. FcRn binding polypeptide according to any one of items        110-121, wherein X_(e) in sequence iii) or v) is D.        123. FcRn binding polypeptide according to any one of items        110-121, wherein X_(e) in sequence iii) or v) is E.        124. FcRn binding polypeptide according to any one of items        110-121, wherein X_(e) in sequence iii) or v) is S.        125. FcRn binding polypeptide according to any one of items        110-119, 121, 123 and 124, wherein X_(d)X_(e) in sequence iii)        or v) is selected from EE, ES, SE and SS.        126. FcRn binding polypeptide according to item 125, wherein        X_(d)X_(e) in sequence iii) or v) is ES.        127. FcRn binding polypeptide according to item 125, wherein        X_(d)X_(e) in sequence iii) or v) is SE.        128. FcRn binding polypeptide according to any one of items        110-127, wherein X_(f) in sequence iii) or v) is A.        129. FcRn binding polypeptide according to any one of items        110-127, wherein X_(f) in sequence iii) or v) is S.        130. FcRn binding polypeptide according to item 110 or 111,        wherein in sequence iii) or v), X_(a) is A; X_(b) is N; X_(c) is        A and X_(f) is A.        131. FcRn binding polypeptide according to item 110 or 111,        wherein in sequence iii) or v), X_(a) is A; X_(b) is N; X_(c) is        C and X_(f) is A.        132. FcRn binding polypeptide according to item 110 or 111,        wherein in sequence iii) or v), X_(a) is S, X_(b) is E; X_(c) is        S and X_(f) is S.        133. FcRn binding polypeptide according to item 110 or 111,        wherein in sequence iii) or v), X_(a) is S, X_(b) is E; X_(c) is        C and X_(f) is S.        134. FcRn binding polypeptide according to item 110 or 111,        wherein in sequence iii) or v), X_(a) is A; X_(b) is N; X_(c) is        A; X_(d)X_(e) is ND and X_(f) is A.        135. FcRn binding polypeptide according to item 110 or 111,        wherein in sequence iii) or v), X_(a) is A; X_(b) is N; X_(c) is        C; X_(d)X_(e) is ND and X_(f) is A.        136. FcRn binding polypeptide according to item 110 or 111,        wherein in sequence iii) or v), X_(a) is S, X_(b) is E; X_(c) is        S, X_(d)X_(e) is ND and X_(f) is S.        137. FcRn binding polypeptide according to item 110 or 111,        wherein in sequence iii) or v), X_(a) is S, X_(b) is E; X_(c) is        C; X_(d)X_(e) is ND and X_(f) is S.        138. FcRn binding polypeptide according to item 110 or 111,        wherein in sequence iii) or v), X_(a) is A; X_(b) is N; X_(c) is        A; X_(d)X_(e) is SE and X_(f) is A.        139. FcRn binding polypeptide according to item 110 or 111,        wherein in sequence iii) or v), X_(a) is A; X_(b) is N; X_(c) is        C; X_(d)X_(e) is SE and X_(f) is A.        140. FcRn binding polypeptide according to item 110 or 111,        wherein in sequence iii) or v), X_(a) is S, X_(b) is E; X_(c) is        S, X_(d)X_(e) is SE and X_(f) is S.        141. FcRn binding polypeptide according to item 110 or 111,        wherein in sequence iii) or v), X_(a) is S, X_(b) is E; X_(c) is        C; X_(d)X_(e) is SE and X_(f) is S.        142. FcRn binding polypeptide according to item 110 or 111,        wherein in sequence iii) or v), X_(a) is A; X_(b) is N; X_(c) is        A; X_(d)X_(e) is ES and X_(f) is A.        143. FcRn binding polypeptide according to item 110 or 111,        wherein in sequence iii) or v), X_(a) is A; X_(b) is N; X_(c) is        C; X_(d)X_(e) is ES and X_(f) is A.        144. FcRn binding polypeptide according to item 110 or 111,        wherein in sequence iii) or v), X_(a) is S, X_(b) is E; X_(c) is        S, X_(d)X_(e) is ES and X_(f) is S.        145. FcRn binding polypeptide according to item 110 or 111,        wherein in sequence iii) or v), X_(a) is S, X_(b) is E; X_(c) is        C; X_(d)X_(e) is ES and X_(f) is S        146. FcRn binding polypeptide according to any one of items 110        and 112-145, wherein sequence iii) is selected from the group        consisting of SEQ ID NO:354-706.        147. FcRn binding polypeptide according to item 146, wherein        sequence iii) is selected from the group consisting of SEQ ID        NO:354-368, SEQ ID NO:370-493 and SEQ ID NO:706.        148. FcRn binding polypeptide according to item 147, wherein        sequence iii) is selected from the group consisting of SEQ ID        NO:354-355 and SEQ ID NO:370-493.        149. FcRn binding polypeptide according to item 148, wherein        sequence iii) is selected from the group consisting of SEQ ID        NO:354-355, SEQ ID NO:370-445, SEQ ID NO:447-456, SEQ ID        NO:458-478 and SEQ ID NO:480-493.        150. FcRn binding polypeptide according to item 147, wherein        sequence iii) is selected from the group consisting of SEQ ID        NO:354-361, SEQ ID NO:366, SEQ ID NO:372-373, SEQ ID NO:376, SEQ        ID NO:381, SEQ ID NO:394, SEQ ID NO:397, SEQ ID NO:418, SEQ ID        NO:423, SEQ ID NO:426, SEQ ID NO:428-430 and SEQ ID NO:706.        151. FcRn binding polypeptide according to item 149 or 150,        wherein sequence iii) is selected from the group consisting of        SEQ ID NO:354, SEQ ID NO:376, SEQ ID NO:381, SEQ ID NO:394, SEQ        ID NO:397, SEQ ID NO:418, SEQ ID NO:426 and SEQ ID NO:428-430.        152. FcRn binding polypeptide according to item 151, wherein        sequence iii) is selected from the group consisting of SEQ ID        NO:354, SEQ ID NO:376, SEQ ID NO:397, SEQ ID NO:418, SEQ ID        NO:428 and SEQ ID NO:430.        153. FcRn binding polypeptide according to item 152, wherein        sequence iii) is selected from the group consisting of SEQ ID        NO:354, SEQ ID NO:376 and SEQ ID NO:428.        154. FcRn binding polypeptide according to item 153, wherein        sequence iii) is SEQ ID NO:354.        155. FcRn binding polypeptide according to any one of items        1-109, which comprises an amino acid sequence selected from:

vii) (SEQ ID NO: 1081) YAK-[BM]-DPSQS SELLX_(c) EAKKL NDSQA P;wherein [BM] is an FcRn binding motif as defined in any one of items1-105 and X_(c) is selected from A, S and C; and

-   -   viii) an amino acid sequence which has at least 94% identity to        a sequence defined by vii).        156. FcRn binding polypeptide according to any one of items        1-109, which comprises an amino acid sequence selected from:

ix) (SEQ ID NO: 1082) FNK-[BM]-DPSQS ANLLX_(c) EAKKL NDAQA P;wherein [BM] is an FcRn binding motif as defined in any one of items1-105 and X_(c) is selected from A and C; and

-   -   x) an amino acid sequence which has at least 94% identity to a        sequence defined by ix).        157. FcRn binding polypeptide according to item 109, which        comprises an amino acid sequence selected from:

(SEQ ID NO: 1083) ADNNFNK-[BM]-DPSQSANLLSEAKKLNESQAPK; (SEQ ID NO: 1084)ADNKFNK-[BM]-DPSQSANLLAEAKKLNDAQAPK; (SEQ ID NO: 1085)ADNKFNK-[BM]-DPSVSKEILAEAKKLNDAQAPK; (SEQ ID NO: 1086)ADAQQNNFNK-[BM]-DPSQSTNVLGEAKKLNESQAPK; (SEQ ID NO: 1087)AQHDE-[BM]-DPSQSANVLGEAQKLNDSQAPK; (SEQ ID NO: 1088)VDNKFNK-[BM]-DPSQSANLLAEAKKLNDAQAPK; (SEQ ID NO: 1089)AEAKYAK-[BM]-DPSESSELLSEAKKLNKSQAPK; (SEQ ID NO: 1090)VDAKYAK-[BM]-DPSQSSELLAEAKKLNDAQAPK; (SEQ ID NO: 1091)VDAKYAK-[BM]-DPSQSSELLAEAKKLNDSQAPK; (SEQ ID NO: 1092)AEAKYAK-[BM]-DPSQSSELLSEAKKLNDSQAPK; (SEQ ID NO: 1093)AEAKYAK-[BM]-DPSQSSELLSEAKKLSESQAPK; (SEQ ID NO: 1094)AEAKYAK-[BM]-DPSQSSELLSEAKKLESSQAPK; (SEQ ID NO: 1095)VDAKYAK-[BM]-DPSQSSELLSEAKKLNDSQAPK; (SEQ ID NO: 1096)VDAKYAK-[BM]-DPSQSSELLSEAKKLSESQAPK; (SEQ ID NO: 1097)VDAKYAK-[BM]-DPSQSSELLSEAKKLESSQAPK; (SEQ ID NO: 1098)VDAKYAK-[BM]-DPSQSSELLAEAKKLNKAQAPK; and (SEQ ID NO: 1099)AEAKYAK-[BM]-DPSQSSELLAEAKKLNKAQAPK;wherein [BM] is an FcRn binding motif as defined in any one of items1-105.158. FcRn binding polypeptide according to any preceding item, whichcomprises an amino acid sequence selected from:

xi) (SEQ ID NO: 1078) AEAKYAK-[BM]-DPSQSSELLSEAKKLNDSQAPK;wherein [BM] is an FcRn binding motif as defined in any one of items1-105; and

-   -   xii) an amino acid sequence which has at least 94% identity to        the sequence defined in xi).        159. FcRn binding polypeptide according to item 158, in which        sequence xi) is selected from the group consisting of SEQ ID        NO:1060-1062.        160. FcRn binding polypeptide according to any preceding item,        which comprises an amino acid sequence selected from:

xiii) (SEQ ID NO: 1079) VDAKYAK-[BM]-DPSQSSELLSEAKKLNDSQAPK;wherein [BM] is an FcRn binding motif as defined in any one of items1-105; and

-   -   xiv) an amino acid sequence which has at least 94% identity to        the sequence defined in xiii).        161. FcRn binding polypeptide according to item 160, in which        sequence xiii) is selected from the group consisting of SEQ ID        NO: 707-1059.        162. FcRn binding polypeptide according to item 161, in which        sequence xiii) is selected from the group consisting of SEQ ID        NO:707-721, SEQ ID NO:723-846 and SEQ ID NO:1059.        163. FcRn binding polypeptide according to item 162, in which        sequence xiii) is selected from the group consisting of SEQ ID        NO:707-708 and SEQ ID NO:723-846.        164. FcRn binding polypeptide according to item 163, in which        sequence xiii) is selected from the group consisting of SEQ ID        NO:707-708, SEQ ID NO:723-798, SEQ ID NO:800-809, SEQ ID        NO:811-831 and SEQ ID NO:833-846.        165. FcRn binding polypeptide according to item 162, in which        sequence xiii) is selected from the group consisting of SEQ ID        NO:707-714, SEQ ID NO:719, SEQ ID NO:725-726, SEQ ID NO:729, SEQ        ID NO:734, SEQ ID NO:747, SEQ ID NO:750, SEQ ID NO:771, SEQ ID        NO:776, SEQ ID NO:779, SEQ ID NO:781-783 and SEQ ID NO:1059.        166. FcRn binding polypeptide according to item 163 or 165, in        which sequence xiii) is selected from the group consisting of        SEQ ID NO:707, SEQ ID NO:729, SEQ ID NO:734, SEQ ID NO:747, SEQ        ID NO:750, SEQ ID NO:771, SEQ ID NO:779 and SEQ ID NO:781-783.        167. FcRn binding polypeptide according to item 166, in which        sequence xiii) is selected from the group consisting of SEQ ID        NO:707, SEQ ID NO:729, SEQ ID NO:750, SEQ ID NO:771, SEQ ID        NO:781 and SEQ ID NO:783.        168. FcRn binding polypeptide according to item 167, in which        sequence xiii) is selected from the group consisting of SEQ ID        NO:707, SEQ ID NO:729 and SEQ ID NO:781.        169. FcRn binding polypeptide according to item 168, in which        sequence xiii) is SEQ ID NO:707.        170. FcRn binding polypeptide according to any preceding item,        which is capable of binding to FcRn at pH 6.0 such that the        K_(D) value of the interaction is at most 1×10⁻⁶ M, such as at        most 1×10⁻⁷ M, such as at most 1×10⁻⁸ M, such as at most 1×10⁻⁹        M, such as at most 1×10⁻¹⁹ M.        171. FcRn binding polypeptide according to any preceding item,        wherein the K_(D) value of the interaction between FcRn binding        polypeptide and FcRn at pH 7.4 is higher than the K_(D) value of        said interaction at pH 6.0, such as at least 2 times higher,        such as at least 5 times higher, such as at least 10 times        higher, such as at least 50 times higher, such as at least 100        times higher than the K_(D) value of said interaction at pH 6.0.        172. FcRn binding polypeptide according to any preceding item,        wherein the K_(D) value of said interaction at pH 7.4 is at        least 1×10⁻⁸ M, such as at least 1×10⁻⁷ M, such as at least        1×10⁻⁶ M, such as at least 1×10⁻⁵ M.        173. FcRn binding polypeptide according to any one of items        1-170, wherein the K_(D) value of said interaction at pH 7.4 is        the same as or lower than the K_(D) value of said interaction at        pH 6.0.        174. FcRn binding polypeptide according to any one of items        1-170, wherein the K_(D) value of said interaction at pH 7.4 is        at most 1×10⁻⁶ M, such as at most 1×10⁻⁷ M, such as at most        1×10⁻⁸ M, such as at most 1×10⁻⁹ M, such as at most 1×10⁻¹⁹ M.        175. FcRn binding polypeptide according to any preceding item,        which comprises at least one additional amino acid at the        C-terminal and/or N-terminal end.        176. FcRn binding polypeptide according to item 175, wherein        said at least one additional amino acid extension improves        production, purification, stabilization in vivo or in vitro,        coupling or detection of the polypeptide.        177. FcRn binding polypeptide according to any preceding item in        multimeric form, comprising at least two FcRn binding        polypeptide monomer units, whose amino acid sequences may be the        same or different.        178. FcRn binding polypeptide according to item 177, wherein        said FcRn binding polypeptide monomer units are covalently        coupled together.        179. FcRn binding polypeptide according to item 177, wherein the        FcRn binding polypeptide monomer units are expressed as a fusion        protein.        180. FcRn binding polypeptide according to any one of items        177-179, in dimeric form.        181. Fusion protein or conjugate comprising    -   a first moiety consisting of an FcRn binding polypeptide        according to any preceding item; and    -   a second moiety consisting of a polypeptide having a desired        biological activity.        182. Fusion protein or conjugate according to item 181, wherein        the in vivo half-life of said fusion protein or conjugate is        longer than the in vivo half-life of the polypeptide having a        desired biological activity per se.        183. Fusion protein or conjugate according to any one of items        181-182, wherein said desired biological activity is a        therapeutic activity.        184. Fusion protein or conjugate according to any one of items        181-182, wherein said desired biological activity is a binding        activity to a selected target.        185. Fusion protein or conjugate according to item 184, wherein        said selected target is albumin.        186. Fusion protein or conjugate according to item 185, wherein        said albumin binding activity is provided by the albumin binding        domain of streptococcal protein G, or a derivative thereof.        187. Fusion protein or conjugate according to any one of items        185-189, wherein said albumin binding activity increases in vivo        half-life of the fusion protein or conjugate.        188. Fusion protein or conjugate according to any one of items        181-182, wherein said desired biological activity is an        enzymatic activity.        189. Fusion protein or conjugate according to any one of items        181-183, wherein the second moiety having a desired biological        activity is a therapeutically active polypeptide.        190. Fusion protein or conjugate according to any one of items        181-183 and 188-189, wherein the second moiety having a desired        biological activity is selected from the group consisting of        enzymes, hormones, growth factors, chemokines and cytokines.        191. FcRn binding polypeptide, fusion protein or conjugate        according to any preceding item, which inhibits binding of IgG        to FcRn.        192. FcRn binding polypeptide, fusion protein or conjugate        according to item 191, wherein the K_(D) value of the        interaction between said FcRn binding polypeptide, fusion        protein or conjugate and FcRn is lower than the K_(D) value of        the interaction between IgG and FcRn.        193. FcRn binding polypeptide, fusion protein or conjugate        according to any preceding item, further comprising a label.        194. FcRn binding polypeptide, fusion protein or conjugate        according to item 193, wherein said label is selected from the        group consisting of fluorescent dyes and metals, chromophoric        dyes, chemiluminescent compounds and bioluminescent proteins,        enzymes, radionuclides and particles.        195. FcRn binding polypeptide, fusion protein or conjugate        according to any preceding item, comprising a chelating        environment provided by a polyaminopolycarboxylate chelator        conjugated to the FcRn binding polypeptide via a thiol group of        a cysteine residue or an amine group of a lysine residue.        196. FcRn binding polypeptide, fusion protein or conjugate        according to item 195, wherein the polyaminopolycarboxylate        chelator is 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic        acid or a derivative thereof.        197. FcRn binding polypeptide, fusion protein or conjugate        according to item 196, wherein the        1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid        derivative is 1,4,7,10-tetraazacyclododecane-1,4,7-tris-acetic        acid-10-maleimidoethylacetamide.        198. FcRn binding polypeptide, fusion protein or conjugate        according to item 195, wherein the polyaminopolycarboxylate        chelator is 1,4,7-triazacyclononane-1,4,7-triacetic acid or a        derivative thereof.        199. FcRn binding polypeptide, fusion protein or conjugate        according to item 195, wherein the polyaminopolycarboxylate        chelator is diethylenetriaminepentaacetic acid or derivatives        thereof.        200. A polynucleotide encoding a polypeptide according to any        one of items 1-192.        201. Expression vector comprising a polynucleotide according to        item 200.        202. Host cell comprising an expression vector according to item        201.        203. Method of producing a polypeptide according to any one of        items 1-192, comprising    -   culturing a host cell according to item 202 under conditions        permissive of expression of said polypeptide from said        expression vector, and    -   isolating said polypeptide.        204. Composition comprising an FcRn binding polypeptide, fusion        protein or conjugate according to any one of items 1-199 and at        least one pharmaceutically acceptable excipient or carrier.        205. Composition according to item 204, further comprising at        least one additional active agent.        206. Composition according to any one of items 204-205, which is        adapted for administration by a route selected from the group        consisting of oral administration, intranasal administration,        pulmonar administration, vaginal administration, rectal        administration, intravenous injection, intraperitoneal        injection, intramuscular injection, subcutaneous injection and        intradermal injection.        207. FcRn binding polypeptide, fusion protein or conjugate        according to any one of items 1-199 or composition according to        any one of items 204-206 for use as a medicament.        208. FcRn binding polypeptide, fusion protein, conjugate or        composition for use according to item 207, wherein said        medicament is intended for treatment of an auto-immune        condition.        209. FcRn binding polypeptide, fusion protein, conjugate or        composition for use according to item 207 or 208, wherein said        medicament is intended for treatment of a condition selected        from the group consisting of myasthenia gravis, Guillain-Barré        syndrome, autoimmune limbic encephalitis, pediatric autoimmune        neuropsychiatric disorders associated with streptococcal        infection (PANDAS), neuromyotonia (Isaac's syndrome), morvan        syndrome, multiple sclerosis, pemphigus vulgaris, foliaceus,        bullous pemphigoid, epidermolysis bullosa acquisita, pemphigoid        gestationis, mucous membrane pemphigoid, lichen sclerosus,        antiphospholipid syndrome, erlapsing polychondritis, autoimmune        anemia, idiopathic trombocytic purpura, autoimmune Grave's        disease, dilated cardiomyopathy, vasculitis, Goodpasture's        syndrome, idiopathic membranous nephropathy, rheumatoid        arthritis and systemic lupus erythematosus.        210. Method of treatment of a subject in need thereof,        comprising administering to the subject a therapeutically active        amount of an FcRn binding polypeptide, fusion protein or        conjugate according to any one of items 1-199 or composition        according to any one of items 204-206.        211. Method according to item 210, for treatment of an        auto-immune condition.        212. Method according to item 210 or 211, wherein said subject        is suffering from a condition selected from the group consisting        of myasthenia gravis, Guillain Barré syndrome, autoimmune limbic        encephalitis, pediatric autoimmune neuropsychiatric disorders        associated with streptococcal infection (PANDAS), neuromyotonia        (Isaac's syndrome), morvan syndrome, multiple sclerosis,        pemphigus vulgaris, foliaceus, bullous pemphigoid, epidermolysis        bullosa acquisita, pemphigoid gestationis, mucous membrane        pemphigoid, lichen sclerosus, antiphospholipid syndrome,        erlapsing polychondritis, autoimmune anemia, idiopathic        trombocytic purpura, autoimmune Grave's disease, dilated        cardiomyopathy, vasculitis, Goodpasture's syndrome, idiopathic        membranous nephropathy, rheumatoid arthritis and systemic lupus        erythematosus.

1. An FcRn binding polypeptide, comprising an FcRn binding motif BM,which motif consists of the amino acid sequence (SEQ ID NO: 1075)EX₂ X₃ X₄ AX₆ X₇ EIR WLPNLX₁₆X₁₇ X₁₈ QR X₂₁ AFIX₂₅ X₂₆LX₂₈ X₂₉

wherein, independently from each other, X₂ is selected from A, D, E, F,H, I, K, L, N, Q, R, S, T, V, W and Y; X₃ is selected from A, D, E, F,G, H, I, K, L, M, N, Q, R, S, T, V, W and Y; X₄ is selected from A, D,E, F, G, H, I, K, L, N, Q, R, S, T, V, W and Y; X₆ is selected from A,E, F, G, H, I, K, Q, R, S and V; X₇ is selected from A, F, H, K, N, Q,R, S and V; X₁₆ is selected from N and T; X₁₇ is selected from F, W andY; X₁₈ is selected from A, D, E and N; X₂₁ is selected from A, S, V andW; X₂₅ is selected from D, E, G, H, I, K, L, N, Q, R, S, T, V, W and Y;X₂₆ is selected from K and S, X₂₈ is selected from A, D, E, F, H, I, K,L, N, Q, R, S, T, V, W and Y; and X₂₉ is selected from D and R.
 2. TheFcRn binding polypeptide according to claim 1, wherein the BM consistsof an amino acid sequence selected from i) (SEQ ID NO: 1076)EX₂ X₃ X₄ AX₆ HEIR WLPNLTX₁₇ X₁₈ QR X₂₁ AFIX₂₅ KLX₂₈ D

wherein, independently from each other, X₂ is selected from A, D, E, F,H, I, K, L, N, Q, R, S, T, V, W and Y; X₃ is selected from A, D, E, G,H, K, L, M, N, Q, R, S, T, V and Y; X₄ is selected from A, D, E, F, G,I, K, L, N, Q, R, S, T, V and Y; X₆ is selected from A, G, K, R, S andV; X₁₇ is selected from F, W and Y; X₁₈ is selected from A, D, E and N;X₂₁ is selected from A, S, V and W; X₂₅ is selected from D, G, H, K, L,N, R, V and W; X₂₈ is selected from A, D, E, H, K, L, N, Q, R, S, T, Wand Y; and ii) an amino acid sequence which has at least 96% identity tosaid sequence.
 3. The FcRn binding polypeptide according to claim 1,wherein sequence i) fulfills at least three of the six conditions I-VI:I. X₆ is selected from A, G, K and S, II. X₇ is H; III. X₁₇ is selectedfrom F and Y; IV. X₁₈ is D; V. X₂₁ is selected from V and W; VI. X₂₅ isselected from H and R.
 4. The FcRn binding polypeptide according toclaim 1, wherein the sequence is selected from the group consisting ofSEQ ID NO:1-353.
 5. The FcRn binding polypeptide according to claim 1,wherein said FcRn binding motif forms part of a three-helix bundleprotein domain.
 6. The FcRn binding polypeptide according to claim 1,which comprises an amino acid sequence selected from: iii)(SEQ ID NO: 1077) K-[BM]-DPSQS X_(a)X_(b)LLX_(c) EAKKL X_(d)X_(e)X_(f)Q;

wherein [BM] is an FcRn binding motif as defined herein, provided thatX₂₉ is D; X_(a) is selected from A and S, X_(b) is selected from N andE; X_(c) is selected from A, S and C; X_(d) is selected from E, N and S,X_(e) is selected from D, E and S, X_(f) is selected from A and S, andiv) an amino acid sequence which has at least 93% identity to a sequencedefined by iii).
 7. The FcRn binding polypeptide according to claim 6,wherein sequence iii) is selected from the group consisting of SEQ IDNO:354-706.
 8. The FcRn binding polypeptide according to claim 1, whichcomprises an amino acid sequence selected from: xi) (SEQ ID NO: 1092)AEAKYAK-[BM]-DPSQSSELLSEAKKLNDSQAPK;

wherein [BM] is an FcRn binding motif as defined in claim 1; and xii) anamino acid sequence which has at least 94% identity to the sequencedefined in xi).
 9. The FcRn binding polypeptide according to claim 8, inwhich sequence xi) is selected from the group consisting of SEQ IDNO:1060-1062.
 10. The FcRn binding polypeptide according to claim 1,which comprises an amino acid sequence selected from: xiii)(SEQ ID NO: 1095) VDAKYAK-[BM]-DPSQSSELLSEAKKLNDSQAPK,

wherein [BM] is an FcRn binding motif as defined in claim 1; and xiv) anamino acid sequence which has at least 94% identity to the sequencedefined in xiii).
 11. The FcRn binding polypeptide according to claim10, in which sequence xiii) is selected from the group consisting of SEQID NO:707-1059.
 12. The FcRn binding polypeptide according to claim 1,which is capable of binding to FcRn at pH 6.0 such that the K_(D) valueof the interaction is at most 1×10⁻⁶ M.
 13. The FcRn binding polypeptideaccording to claim 1, wherein the K_(D) value of the interaction betweenFcRn binding polypeptide and FcRn at pH 7.4 is higher than the K_(D)value of said interaction at pH 6.0.
 14. The FcRn binding polypeptideaccording to claim 1, wherein the K_(D) value of the interaction betweenFcRn binding polypeptide and FcRn at pH 7.4 is at least 1×10⁻⁸ M. 15.The FcRn binding polypeptide according to claim 1, wherein the K_(D)value of the interaction between FcRn binding polypeptide and FcRn at pH7.4 is at most 1×10⁻⁶ M.
 16. A fusion protein or conjugate comprising afirst moiety consisting of an FcRn binding polypeptide according toclaim 1; and a second moiety consisting of a polypeptide having adesired biological activity.
 17. An FcRn binding polypeptide, fusionprotein or conjugate according to claim 1, which inhibits binding of IgGto FcRn.
 18. A polynucleotide encoding a polypeptide according toclaim
 1. 19. A composition comprising an FcRn binding polypeptide,fusion protein or conjugate according to claim 1 and at least onepharmaceutically acceptable excipient or carrier.
 20. An FcRn bindingpolypeptide, fusion protein or conjugate according to claim 1 for use asa medicament.